Illumina Bio-Rad SureCell WTA 3' Library Prep for Peripheral Blood Mononuclear Cells (PBMC)

Transcription

1 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Demonstrated Protocol Docment # v00 December 2017 For Research Use Only. Not for se in diagnostic procedres. ILLUMINA PROPRIETARY

2 Table of Contents Chapter 1 Overview 1 Introdction 1 Additional Resorces 1 Chapter 2 Protocol 3 Introdction 3 Critical Workflow Steps 4 Tips and Techniqes 4 Preliminary Cell Prep Optimization Gidelines 5 Library Prep Workflow 7 Prepare, Cont, and Assess Viability of Single-Cell Sspension 8 Prepare Cell and Barcode Sspension Mixes 9 Isolate Single Cells 12 Reverse Transcribe Samples 19 Break Emlsion 20 Clean Up First Strand Synthesis 21 Synthesize Second Strand cdna 25 Clean Up cdna 26 Tagment cdna 29 Amplify Tagmented cdna 30 Clean Up Libraries 31 Assess Libraries 34 Prepare for Seqencing 35 Appendix A Spporting Information 37 Introdction 37 How does the SreCell WTA 3' Assay Work? 37 Cell Conting Protocol 37 Lab Tracking Chart 39 Acronyms 40 Kit Options 40 Consmables and Eqipment 43 Technical Assistance 44 Docment # v00 For Research Use Only. Not for se in diagnostic procedres. ii

3 Chapter 1 Overview Introdction 1 Additional Resorces 1 Introdction This protocol describes how to prepare 3 -tagged RNA-Seq libraries from peripheral blood mononclear cells (PBMC) for whole transcriptome gene profiling analysis on Illmina seqencing systems. The protocol reqires a Bio-Rad ddseq Single-Cell Isolator and reagents provided in the Illmina Bio-Rad SreCell WTA 3' Library Prep Kit for the ddseq System to isolate single cells and barcode individal transcriptomes. The SreCell WTA 3' demonstrated protocol for PBMC is optimized for 1000 single cells as otpt for each ddseq Single-Cell Isolator cartridge. Each cartridge has for chambers that can be loaded with p to two niqe samples for an average cell otpt of 500 cells per sample. Typical genic cont for PBMCs are ~400 genes per cell. For more information on optimal cell nmber recommended for tertiary analysis, see the Single-Cell RNA Seqencing of Peripheral Blood Mononclear Cells Technical Note. Libraries generated from this demonstrated protocol have been evalated on the NextSeq platform. A representative data set can be fond on BaseSpace Seqence Hb Pblic Data. Performance on other Illmina platforms may reqire additional ser optimization. The SreCell WTA 3 Library Prep Kit for the ddseq System protocol incldes the following featres: Comprehensive workflow for single-cell analysis of 3' RNA transcripts Significant redction in the time from cell cltre to cell lysis sing the ddseq Single-Cell Isolator for cell isolation Individal droplets have cell lysis, cell barcoding, and niqe molecle tagging 15 minte tagmentation process to fragment cdna and add adapter seqences Benefits of sing master mixed reagents, saving reagent containers, pipetting, and hands-on time Data analysis is condcted sing the Illmina SreCell RNA Single-Cell App in the BaseSpace Seqence Hb. The SreCell RNA Single-Cell App performs sample demltiplexing, single-cell identification, genome alignment, 3 gene conting, and cell clstering. The SreCell RNA Single-Cell App spports mltiple species, inclding, bt not limited to, hman, mose, and rat genomes. DISCLAIMER The information in this Illmina Demonstrated Protocol is being provided as a cortesy. In some cases, reagents are reqired to be prchased from non-athorized third-party sppliers. Illmina does not garantee or promise technical spport for the performance of or prodcts sed with any reagent prchased from a non-athorized third-party spplier. Additional Resorces Visit the SreCell WTA 3 Library Prep Kit for the ddseq System spport page on the Illmina website or ddseq Single-Cell Isolator page on the Bio-Rad website for docmentation, software downloads, training resorces, and information abot compatible Illmina and Bio-Rad prodcts. The following docmentation is available for download from the Illmina website. Resorce Cstom Protocol Selector Consmables and Eqipment List (docment # ) Description A wizard for generating cstomized end-to-end docmentation that is tailored to the library prep method, rn parameters, and analysis method sed for the seqencing rn. Provides an interactive checklist of ser-provided consmables and eqipment. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 1

4 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) The following docmentation is available for download from the Bio-Rad website. Resorce ddseq Single-Cell Isolator Instrction Manal (Docment # ) Description Provides instrctions for installing and operating the Bio-Rad ddseq Single-Cell Isolator. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 2

5 Chapter 2 Protocol Introdction 3 Critical Workflow Steps 4 Tips and Techniqes 4 Preliminary Cell Prep Optimization Gidelines 5 Library Prep Workflow 7 Prepare, Cont, and Assess Viability of Single-Cell Sspension 8 Prepare Cell and Barcode Sspension Mixes 9 Isolate Single Cells 12 Reverse Transcribe Samples 19 Break Emlsion 20 Clean Up First Strand Synthesis 21 Synthesize Second Strand cdna 25 Clean Up cdna 26 Tagment cdna 29 Amplify Tagmented cdna 30 Clean Up Libraries 31 Assess Libraries 34 Prepare for Seqencing 35 Introdction This chapter describes the SreCell WTA 3 Library Prep Kit for the ddseq Systemprotocol, from cell preparation throgh qalification and qantification of final libraries for seqencing. Before yo begin, do the following. Review SreCell Best Practices on the Illmina website. Confirm that Illmina Experiment Manager v1.13 or later is sed to set p the seqencing sample sheet if BaseSpace Prep Tab is not sed. Version 1.13 or later has the appropriate UMI settings and index seqences for sample demltiplexing. When sing BaseSpace Seqence Hb for seqencing analysis, confirm that SreCell RNA Single Cell App v1.2 or later is sed. Confirm that bcl2fastq v2.18 or later is sed for FASTQ generation. This protocol is verified to process p to for cartridges in one experiment. If this is yor first experiment, process 1 2 cartridges. If yo are processing more than for cartridges, contact Illmina Tech Spport for a modified protocol. Confirm that the ddseq Single-Cell Isolator is installed and operating properly. Review pipetting techniqes in the ddseq Single-Cell Isolator Instrction Manal. Confirm kit contents and make sre that yo have the reqired eqipment and consmables. This protocol reqires two different magnetic stands dring library clean-p procedres. See Spporting Information on page 37. Review the color-coded caps that identify the associated sspension mix of the reagents in this protocol. Red caps identify reagents sed to create cell enzyme mix Ble caps identify reagents sed to create barcode sspension mix Use a Lab Tracking Chart to record sample observations throghot the protocol. See Lab Tracking Chart on page 39. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 3

6 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Critical Workflow Steps Several steps within the workflow reqire additional attention and are key to single-cell library sccess. 1 Filter cells throgh a cell strainer with sfficient pore size to remove cell clmps. 2 Visally confirm that cells are dissociated to single-cell sspension. 3 Keep yor cell sspensions on ice at all times. 4 A bead prification step prifies single-cell DNA from sample wells containing separate oil and aqeos layers. Visally confirm that magnetic beads are well-mixed. 5 A cstom seqencing primer is provided for Read 1. Dilte the provided primer to the concentration specified for the seqencer yo are sing. Tips and Techniqes Unless a safe stopping point is specified in the protocol, proceed immediately to the next step. Designating Separate Areas Condct all tisse and cell activities in a designated aseptic area that is restricted to cell cltre work. Condct all pre-pcr activities (cell lysis, tagmentation, and amplification preparation procedres) in a dedicated environment physically separated from amplified genetic material (post-pcr). Do not pass material or eqipment from the post-pcr area to the pre-pcr area. Conslt yor local reglations for niversal precations regarding amplicon control practices and biohazardos material handling. HUMAN MATERIAL PRECAUTION Use cation when handling any hman cells. Hman PBMC shold be handled as if capable of transmitting infectios agents. Use niversal precations as recommended by the CDC/NIH manal Biosafety in in Microbiological and Biomedical Laboratories (BMBL) 5th Edition. Avoiding Cross-Contamination When adding or transferring samples, change tips between each sample. Use aerosol-resistant pipette tips to redce the risks of reagent carry-over and sample-to-sample crosscontamination. Sealing the Plate Always seal the 96-well plate before the following steps in the protocol: Centrifge steps Thermal cycling steps Use 8-strip tbe caps to seal plates. Handling Prification (Magnetic) Beads This protocol does not inclde excess Prification Beads (SPB) reagent volme for dispensing from a reservoir and discarding excess volme. Use a single-channel pipette to transfer SPB from the reagent tbe to individal sample wells. Use beads at room temperatre. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 4

7 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Vortex immediately before se. Confirm that the beads are well-dispersed and the color appears homogeneos. Pipette accrate bead volme as this is essential to protocol sccess: Beads are viscos; pipette beads slowly from stock tbe to obtain fll volme. Remove any additional drops collected on the otside of the tip before dispensing to the sample plate. Dispense beads slowly into the sample plate, allowing time to ensre the entire volme has been dispensed from the pipette tip. Do not prime pipette tips with bead soltion. When washing beads: Always prepare fresh 80% ethanol for wash steps. Ethanol can absorb water from the air, impacting yor reslts. Use the specified magnet for the plate. Dispense liqid so that beads on the side of the wells are wetted. Keep the plate on the magnet ntil the instrctions specify to remove it. Do not agitate the plate while on the magnetic stand. Do not distrb the bead pellet. If beads are aspirated into the pipette tips, dispense them back to the plate on the magnetic stand and wait a few mintes ntil the liqid is clear. Do not let the beads dry ot nless specified by the protocol. Preliminary Cell Prep Optimization Gidelines CAUTION Before starting the protocol, make sre yo have the specified cell qantities, consmables, and eqipment reqired to complete the protocol. After cells have been prepared, there are no safe stopping points ntil Synthesize Second Strand cdna has begn. Proceed immediately to each step in the protocol. Cells mst be kept cold on ice at all times bt shold not be frozen. Do not remove the cells from ice ntil instrcted to do so dring Isolate Single Cells on page 12. The following attribtes are critical for the sccess of the SreCell WTA 3' assay. PBMC sorce This protocol has been developed sing same-day prepared PBMCs isolated by a Ficoll gradient prification method. Other PBMC isolation methods, inclding cell sorting, have not been evalated. In addition, freshly frozen PBMCs have been sccessflly tested sing this protocol. For more information, see the Single-Cell RNA Seqencing of Peripheral Blood Mononclear Cells Technical Note. PBMCs stored nder other conditions may reqire frther testing. Flly dissociated single cells Single-cell encapslation by the ddseq Single-Cell Isolator instrment reqires single cell sspension as inpt. Cell aggregates or doblets present in the sspension will significantly increase the probability of doblets or mltiplets dring single-cell isolation on the ddseq, making data interpretation potentially more difficlt. Depending on the method or cell conter device sed, mltiplets also can affect the accracy of cell conting. While PBMCs rarely form doblets or clmps, perform a visal check nder a microscope to confirm single cell stats before proceeding to encapslation. Accrate cellcont Accrate cellcont is critical to achieve target cellthroghpt and to avoid cell mtiplets. Illmina and Bio-Rad have validated an atomated cell conter (Bio-Rad TC20) for cell conting of PBMC cells. Size-based gating for atomated conters or manal cont may be reqired to avoid conting cell debris. Both viable and non-viable cells shold be inclded in total cell cont. To ensre the accracy of the PBMC cell cont, the PBMC sspension shold be free of red blood cells. The presence of red blood cells is indicated by a red or pink color of the pelleted PBMC. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 5

8 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) High viability (>80%) and integrity Dead or damaged cells can release ncleic acids into the cell sspension bffer. This backgrond signal from these cells remains throgh sbseqent steps, and may impact the qality of the reslting analysis. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 6

9 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Library Prep Workflow Figre 1 SreCell WTA 3 Library Prep Kit for the ddseq System Workflow Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 7

10 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Prepare, Cont, and Assess Viability of Single-Cell Sspension This section describes preparation of a single-cell sspension, conting of cells, and assessment of cell viability. The SreCell WTA 3' PBMC demonstrated protocol is optimized for a single-cell sspension inpt of 3000 cells/µl at greater than 80% viability with a minimm of 2500 c/µl. All for chambers of a cartridge mst be loaded with cells. Up to two niqe samples can be processed per cartridge. Each sample chamber reqires 250,000 cells for processing. The expected otpt is ~500 cells per sample (or ~1000 cells per cartridge if loaded with the same sample). After cells have been prepared, there are no safe stopping points ntil the Second Strand Synthesis has started. Proceed immediately to each step in the protocol. CAUTION Delays dring cell preparation and handling can lead to sample failre. Make sre yo have all reqired consmables (see Consmables and Eqipment on page 43) before yo begin. Do not stop dring or between steps. Consmables BSA (Bovine Serm Albmin) PBS (Phosphate-Bffered Saline) Trypan ble Hemacytometer or slides for cell conting Rainin pipettes Gidelines P200 single channel or P1000 single channel Review Preliminary Cell Prep Optimization Gidelines on page 5 before yo begin. Preparation Prepare 1X PBS + 0.1% and store on ice. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 8

11 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Procedre 1 Wash cells twice in cold 1X PBS + 0.1% BSA at a volme sfficient to remove carryover components from the media. 2 Centrifge for 5-10 mintes at 250 x g and resspend cells in the appropriate volmeof cold 1X PBS + 0.1% BSA3000 cells/µl. Keep cells on ice. 3 Use a microscope or atomated cell conter imaging featre to assess cell viability and concentration. 4 Dilte the stock cell preparation to target 3000 cells/µl in 1XPBS +0.1% BSA soltion. Keep dilted cells on ice ntil se. CAUTION Cell concentration below 2500 cells/µl can adversely affect assay performance. 5 Proceed to Prepare Cell and Barcode Sspension Mixes on page 9 immediately after preparing the single-cell sspension. Single-cell sspension can remain on ice for p to one hor before loading on the ddseq Single-Cell Isolator. Figre 2 PBMC at 3000 cells/µl Prepare Cell and Barcode Sspension Mixes This step prepares sspension mixes that add first strand synthesis components before loading on the ddseq Single-Cell Isolator. Cell sspension mix incldes all the reagents necessary to perform the first strand synthesis (RT) from the messenger RNA released from the single cell in the droplet after cell lysis. Barcode sspension mix contains the barcoded beads and UMI (niqe moleclar identifier) elements that allow specific tagging of messenger RNA. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 9

12 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) CAUTION Consmables Delays dring cell preparation and handling can lead to sample failre. Do not stop dring or between steps. Cell Sspend Bffer [red cap] DTT [red cap] RNA Stabilizer [red cap] RT Enzyme [red cap] Enhancer Enzyme [red cap] Barcode Bffer [ble cap] 3' Barcode Mix [ble cap] Rainin pipettes P20 single channel and P200 single channel (se in Procedre on page 11) Preparation 1 Prepare the following consmables: Item Storage Instrctions Cell Sspend Bffer -25 C to -15 C Thaw on ice. Vortex vigorosly to mix, and then centrifge briefly. DTT -25 C to -15 C Thaw at room temperatre. Vortex to mix, then centrifge briefly. RNA Stabilizer -25 C to -15 C Gently invert the thawed tbes 3 5 times, and then centrifge briefly. RT Enzyme -25 C to -15 C Gently invert the thawed tbes 3 5 times, and then centrifge briefly. Enhancer Enzyme -25 C to -15 C Flick the thawed tbes 3 5 times, and then centrifge briefly. Barcode Bffer -25 C to -15 C Thaw on ice. Vortex vigorosly to mix, and then centrifge briefly. 3' Barcode Mix 2 C to 8 C Gently invert the tbe 3 5 times. Keep on ice. Vortex to mix before se. Encapslation Oil 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Invert the tbe five times to mix. This will be sed in Isolate Single Cells on page 12. ddseq Priming Soltion 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. Vortex well to mix, then centrifge briefly. This will be sed in Isolate Single Cells on page 12. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 10

13 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Procedre 1 Create the cell enzyme mix by combining the following components (red caps) in a 1.7 ml tbe on ice. Pipette the cell enzyme mix with a P200 single channel pipette times while on ice, and then centrifge briefly. NOTE It is normal to see bbbles after mixing and centrifgation. Cell Enzyme Mix Component Cell Sspend Bffer 60 DTT 8 RNA Stabilizer 6 RT Enzyme 13.2 Enhancer Enzyme 12 Total 99.2 Volme (µl) for 1 Cartridge (2 Samples) 2 Create the cell sspension mix for each cell type by combining the following components in a new 1.7 ml tbe on ice. Before adding the filtered cells to the cell enzyme mix, vortex the cells for one second, and repeat three times. a b c If loading two niqe samples, make a single sample cell sspension mix for each cell type. To load the same cell sample across all for chambers, make a cell sspension mix sing the volmes listed for one cartridge. All for sample chambers mst be loaded with cell sspension mix. If yo choose not to load any cells into a chamber, prepare and load the cell sspension mix, sbstitting an eqivalent volme 1X PBS + 0.1% BSA in place of cells. Cell Sspension Mix Component Volme (µl) for 1 Sample (2 Chambers) Celli Enzyme Mix Cells (>2500 cells/μl) 9 18 NOTE Volme (µl) for 1 Cartridge (2 Samples) Proceed immediately to the next step. Do not mix the combined components ntil Load Cartridge on page Create the barcode sspension mix by combining the following components (ble caps) in a new 1.7 ml tbe on ice. Before combining, resspend the 3' Barcode Mix by vortexing for one second, repeat three times, and immediately add to the Barcode Bffer. Barcode Sspension Mix Component Barcode Bffer 60 3' Barcode Mix 60 NOTE Volme (µl) for 1 Cartridge Proceed immediately to the next step. Do not mix the combined components ntil Load Cartridge on page 15. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 11

14 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Isolate Single Cells This step ses the ddseq Single-Cell Isolator to coencapslate cells (samples) and barcodes in droplets that create a highly parallelized library prep for single-cell analysis. CAUTION Consmables Delays dring cell preparation and handling can lead to sample failre. Do not stop dring or between steps. 96-well cooling block ddseq cartridge ddseq cartridge holder Rainin pipettes P20 single channel and mltichannel, P50 mltichannel, P200 single channel and mltichannel (se in Prime Cartridge on page 14, Load Cartridge on page 15, and Transfer Samples on page 18) Encapslation Oil ddseq Priming Soltion Bio-Rad ddpcr plate (Bio-Rad catalog # ) 8-tbe strip (General Lab Spplier) 8-tbe strip caps (Bio-Rad, catalog # TCS-0803) Mltichannel Pipette Reservoir Gidelines Make sre the ddseq Single-Cell Isolator is installed according to manfactrer instrctions and the power indicator is lit. Review pipetting gidelines in the ddseq Single-Cell Isolator Instrction Manal. Use Rainin pipettes and corresponding tips to load the cartridge. Use of other tips can negatively impact ddseq cartridge performance. Make sre that the ddseq cartridge is in the cartridge holder when loading reagents. Avoid static generation while handling encapslated samples. Work in a clear, static-free area. Do not se latex gloves when making or handling droplets. Abot Reagents To avoid bbbles, depress the pipette plnger only to the first stop when loading the cartridge. Aspirate and dispense Encapslation Oil slowly de to the viscosity of the soltion. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 12

15 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Preparation Prepare Cartridge 1 Grip the cartridge by the tab and remove it from the package. Do not toch the wells or gaskets. Figre 3 ddseq Cartridge 2 Insert the cartridge into the cartridge holder. a b c Lift the cartridge holder lever. Orient the green gasket on the cartridge with the green stripe on the cartridge holder, insert the tab nder the rails, then slide the cartridge into the holder. Check that the cartridge is flly inserted and lying flat against the bottom of the holder, then close the lever. If the lever does not close completely, reinsert the cartridge. Figre 4 Insert Cartridge Into Cartridge Holder Figre 5 Incorrectly Assembled Cartridge and Cartridge Holder A B Cartridge not flly inserted Cartridge oriented incorrectly Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 13

16 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 6 Assembled Cartridge and Cartridge Holder A B C D Encapslated sample otpt wells Barcode Sspension Mix inpt wells (Ble) Cell Sspension Mix inpt wells (Red) Encapslation Oil inpt wells Procedre Prime Cartridge Prime the ddseq cartridge to prepare flidics for single cell isolation. 1 Use a P200 single channel pipette to add 25 µl of ddseq Priming Soltion to each well of an 8-tbe strip. 2 Use a P20 mltichannel pipette to add 20 µl of ddseq Priming Soltion from the 8-tbe strip to each well of the second row of the cartridge as shown in Figre 7. NOTE Use a mltichannel pipette to avoid missing wells dring cartridge priming. Figre 7 ddseq Priming Soltion Wells A ddseq Priming Soltion wells Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 14

17 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) 3 Allow the ddseq Priming Soltion to remain in wells for one minte, then remove with a P20 mltichannel pipette set to 20 µl. Do not allow the ddseq Priming Soltion to remain in wells longer than three mintes. CAUTION Load Cartridge ddseq Priming Soltion interferes with single cell isolation. Make sre all ddseq Priming Soltion is removed from the wells. Proper mixing of the barcode sspension mix and cell sspension mix provides even distribtion into encapslated droplets. 1 Vortex the barcode sspension mix for one second, and repeat three times. 2 Using a P20 single channel pipette, load 20 μl of the barcode sspension mix into the bottom of the B ports (Ble). Depress the pipette plnger only to the first stop to avoid bbbles. Figre 8 Barcode Sspension Mix Wells 3 Vortex the cell sspension mix for one second, and repeat three times to create a homogeneos single cell sspension. 4 Using a P20 single channel pipette, load 20 µl of cell sspension mix into the bottom of the red ports, nmbered 1 4 if loading one sample. If loading two distinct samples, se ports 1 2 for sample 1 and ports 3 4 for sample 2. Do not se an intercalate sample loading scheme. Depress the pipette plnger only to the first stop to avoid bbbles. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 15

18 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 9 Cell Sspension Mix Wells for two distinct samples 5 Por the Encapslation Oil into a mltichannel pipette reservoir. Using a P200 mltichannel pipette, load 80 µl of Encapslation Oil into each well of the bottom row of the cartridge labeled OIL. Depress the pipette plnger only to the first stop to avoid bbbles. NOTE One bottle of Encapslation Oil is enogh for two cartridges. CAUTION Failre to load the Encapslation Oil will reslt in single cell isolation failre and samples and reagents cannot be recovered. 6 Keep the loaded cartridge in the cartridge holder for single cell isolation on the ddseq Single-Cell Isolator. Generate Single Cell Droplets 1 Press the silver btton on the top of the ddseq Single-Cell Isolator to open the instrment. Figre 10 Bio-Rad ddseq Single-Cell Isolator 2 Place the cartridge holder into the instrment. Make sre that the cartridge indicator light is solid green to confirm that the cartridge holder is in the correct position. If the cartridge indicator light is not lit, reseat the cartridge holder on the magnetic plate. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 16

19 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 11 ddseq Cartridge Loaded on ddseq Single-Cell Isolator 3 Press the silver btton on the top of the ddseq Single-Cell Isolator to close the instrment. Single-cell isolation begins atomatically after the ddseq Single-Cell Isolator door is closed and takes approximately five mintes. The droplet indicator flashes green to indicate that cell isolation is in progress. Singlecell isolation is complete when all three indicator lights are solid green. CAUTION Do not proceed ntil all three indicator lights are solid green. 4 Press the silver btton on the front of the ddseq Single-Cell Isolator to open the instrment. NOTE After the door opens, the instrment contines to make noise for ~five seconds while it resets. 5 Remove the cartridge holder from the ddseq Single-Cell Isolator. Sccessflly encapslated samples appear clody in the otpt wells. Check for wells that look clear or empty, as droplet generation may have failed. Note clear or empty wells in the Lab Tracking Chart on page 39. Figre 12 Encapslated Samples in Otpt Wells A Otpt wells NOTE Proceed immediately to the next step. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 17

20 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Transfer Samples Encapslated samples are transferred to a 96-well cooling block and kept cold ntil starting reverse transcription. Keeping the encapslated samples cold at this step ensres stability of barcoded RNA and encapslated droplets. 1 Chill a 96-well plate by secrely placing it on a chilled 96-well cooling block. NOTE Avoid static generation while handling encapslated samples. 2 Use a P50 mltichannel pipette set at 43 μl to gently and slowly aspirate all encapslated sample from the otpt wells. Fast or harsh pipetting will break the encapslated samples. Pipette very slowly to avoid yield loss. CAUTION Using a single channel pipette to individally transfer encapslated samples will reslt in neven sample volmes. Figre 13 Emlsion Layers A B C Aqeos layer Oil layer Oil + air bbbles NOTE The total emlsion volme transferred to each well is μl and ~5 μl of air. 3 Dispense the encapslated sample as follows. a b c Very slowly dispense the encapslated sample into the corresponding colmn of the plate, as shown in Figre 14. Dispense shold take approximately five seconds. CAUTION Do not discard tips ntil all of the encapslated sample has been transferred to the plate. Discarding tips with sample will reslt in yield loss. Wait five seconds for remaining encapslated sample to collect at the tip of the pipette. Slowly dispense the remaining encapslated sample into the same colmn of the plate. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 18

21 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 14 Transferring Encapslated Samples (two niqe samples) 4 Cover sample wells sing an 8-tbe strip cap and keep samples on the 96-well cooling block ntil Reverse Transcribe Samples on page 19. CAUTION Plastic plate seals can generate static and impact encapslated samples. Use 8-tbe strip caps to seal wells. Other plate seals may generate static and adversely affect encapslated samples. 5 If yo are processing a second cartridge, proceed to Prepare Cartridge on page 13. Add encapslated samples from each additional cartridge to a new colmn of the same 96-well plate. NOTE If yo are processing more than for cartridges, contact Illmina Technical Spport for a modified protocol. 6 If yo have finished processing cartridges, proceed to Reverse Transcribe Samples on page 19. Liqid remaining in the inpt wells after droplet generation is de to flshing sample from the inpt wells this is not left over sample. When removing the cartridge from the cartridge holder, do not invert the cartridge. Dispose of cartridges according to standard laboratory procedres. Reverse Transcribe Samples This step reverse transcribes samples on a thermal cycler. Gidelines Keep the plate on the 96-well cooling block while transporting to the thermal cycler. Work in a clear, static-free area, and avoid static generation while handling encapslated samples. Preparation 1 Save the following Reverse Transcription (RT) program on a thermal cycler: Choose the preheat lid option and set to 105 C Set the reaction volme to 50 µl 37 C for 30 mintes 50 C for 60 mintes 85 C for five mintes Hold at 4 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 19

22 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Procedre 1 Place the 96-well plate on the thermal cycler and rn the RT program. CAUTION Do not vortex or spin down the plate before placing on the thermal cycler. 2 Remove Prification Beads (SPB) from storage and bring to room temperatre for the Clean Up First Strand Synthesis on page 21. Break Emlsion This step breaks the individal droplets containing barcoded sample cells for frther sample processing. Consmables Droplet Disrptor Nclease-free water Rainin pipettes P20 single channel and P200 single channel Preparation 1 Prepare the following consmables: Item Storage Instrctions Droplet Disrptor 2 C to 8 C Vortex 3 5 times immediately before se to mix, then centrifge briefly. This reagent can be kept at room temperatre dring se. Procedre 1 Remove the 96-well plate from the thermal cycler. CAUTION Do not vortex or spin down the plate after removing it from the thermal cycler. 2 Visally examine the samples which shold all have eqal volmes. Each sample has two distinct layers, an oil layer on the bottom and an aqeos layer on top. Note if any wells have only one layer in the Lab Tracking Chart on page Remove the 8-tbe strip caps careflly to avoid cross-sample contamination. 4 Add 20 µl of Droplet Disrptor by dispensing slowly against the side of the well above each sample. Do not mix or pipette Droplet Disrptor into the sample. 5 Wait 30 seconds, then add 100 µl of water by dispensing against the side of the well above each sample. Do not mix or pipette water into the sample. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 20

23 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 15 Sample Emlsion Layers A B Emlsion layer Oil layer NOTE Proceed immediately to the next step. Clean Up First Strand Synthesis This step ses Prification Beads (SPB) to prify the first strand prodct (library cdna), provides a selection step that removes short fragments (nbond barcodes), and combines the two otpt volmes from each sample into a single well. Consmables Resspension Bffer (RSB) Prification Beads (SPB) Freshly prepared 80% ethanol (EtOH) Pipettes P20 single channel and P200 single channel and mltichannel 96-well plate seal Eqipment Magnetic peg stand (Thermo Fisher, catalog # AM10027) DynaMag 96 Side Magnet (Thermo Fisher, catalog # 12331D) or the DynaMag 96 Side Skirted Magnet (Thermo Fisher catalog # 12027) Abot Reagents See Handling Prification (Magnetic) Beads on page 4 for details abot working with Prification Beads (SPB). Abot Samples Sample wells contain separate oil and aqeos layers dring this step. When mixing, mix only in the specified layer. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 21

24 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Abot Magnets This procedre ses two types of magnetic stands. Both are needed in this protocol and are not interchangeable. Magnet Type Illstration Step Procedre Magnetic Peg Stand Use this magnet with Bind on page 22 and Wash on page 24. DynaMag 96 Side Magnet or DynaMag 96 Side Skirted Magnet Use either DynaMag magnet with Elte on page 24. Gidelines Use a single channel pipette to transfer Prification Beads (SPB) to sample wells. Using a mltichannel pipette reservoir and a mltichannel pipette reslts in inadeqate Prification Beads (SPB) reagent volme needed to complete this protocol. This process reqires both a magnetic peg stand and either DynaMag side magnet. Preparation 1 Prepare the following consmables: Item Storage Instrctions Resspension Bffer (RSB) 2 C to 8 C Can be sed after removing from 2 C to 8 C. Do not discard ntil the protocol is complete. Prification Beads (SPB) 2 C to 8 C Let stand for 15 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. Procedre Bind 1 Vortex Prification Beads (SPB) ntil well-dispersed. 2 Use a P200 single channel pipette to add 90 µl Prification Beads (SPB) to the samples by dispensing slowly above the aqeos layer withot mixing. Do not dispense into the oil layer at the bottom of the well. 3 Use a P200 single channel pipette, set to 50 µl, to pipette mix Prification Beads (SPB) in the aqeos layer only ntil the layer is evenly distribted (10-15 times). After mixing, the samples have two distinct layers: an oil layer on the bottom of the well and a homogeneos brown aqeos layer on the top. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 22

25 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) 4 Lift the plate to examine the qality of mix for the aqeos layer closely. CAUTION The aqeos layer shold not appear clear at the top. If parts of the aqeos layer still appear clear or a lighter brown, contine to mix ntil the entire aqeos layer is homogenosly brown. Figre 16 Mixing States From Initial State to Properly Mixed State A B C Initial state with a clear aqeos layer at the top Not properly mixed, indicated by a lighter brown aqeos layer at the top Properly mixed with an entirely homogenos brown aqeos layer Figre 17 Mixed Aqeos Layer and Oil Layer A B Mixed aqeos layer Oil layer 5 Incbate at room temperatre for 10 mintes. 6 Place on a magnetic peg stand and wait 10 mintes. Use a magnetic peg stand ntil Elte on page 24. NOTE The liqid might not be completely clear of beads de to retention of beads in the aqeos and oil layers. 7 Use a P200 single channel pipette, set to 200 µl, to remove and discard all spernatant from each well. Use a fresh pipette tip to go into the well again to discard approximately µl more of spernatant. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 23

26 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Wash 1 Wash two times as follows. a b c d Add 200 µl freshly prepared 80% EtOH to each well. Incbate on the magnetic peg stand for 30 seconds. Remove and discard all spernatant from each well. Repeat steps a - c to wash again. 2 Seal the plate and centrifge at 280 g for 10 seconds to bring down any ethanol or liqid remaining on sides of wells. 3 Place on a magnetic peg stand and wait 30 seconds. 4 Use a P20 single channel pipette to remove residal 80% EtOH from each well. 5 Air-dry on the magnetic peg stand for five mintes. Elte 1 Remove the sample plate from the magnetic peg stand. 2 Use a P20 single channel pipette to add 18 μl Resspension Bffer (RSB) to each sample well. Pipette to mix, making sre all beads are resspended. CAUTION Yield loss can occr if beads are not thoroghly resspended. 3 Incbate at room temperatre for two mintes. 4 Seal the plate and centrifge at 280 g for 10 seconds. 5 Place on a DynaMag 96 side magnet and wait two mintes. NOTE Yo may proceed to the next step even if the soltion is not completely clear after two mintes. Combine Wells from Sample and Transfer 1 Using a P20 single channel pipette, combine the for wells for each sample into a single well by transferring 17 µl of spernatant from each sample well to a new plate, as follows. Keep the sample plate on the DynaMag 96 side magnet dring this step. Sample 1, rows A D to row A of the corresponding colmn in the new plate. Sample 2, rows E H to row B of the corresponding colmn in the new plate. After transferring, the total volme of spernatant in each well of the new plate is 68 µl. CAUTION Each for sample wells in sccession represent one sample. Proper pooling is critical for library prep indexing and sample processing. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 24

27 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 18 Combining Barcoded Samples (two niqe samples) NOTE Proceed immediately to the next step. Synthesize Second Strand cdna This process removes the RNA template and synthesizes a replacement strand to generate doble stranded cdna. Consmables Second Strand Bffer (SSB) Second Strand Enzyme (SSE) Pipettes P20 single channel and P200 mltichannel Abot Reagents Second Strand Enzyme (SSE) is viscos and pipettes slowly. Ensre that the specified volme is obtained. Preparation 1 Prepare the following consmables: Item Storage Instrctions Second Strand Bffer (SSB) -25 C to -15 C Thaw on ice. Vortex to mix, and then centrifge briefly. Keep on ice ntil se. Second Strand Enzyme (SSE) -25 C to -15 C Thaw on ice. Pipette mix and then centrifge briefly. Keep on ice ntil se. 2 Save the following Second Strand Synthesis (SSS) program on the thermal cycler: Trn off the heated lid fnction Set the reaction volme to 80 μl 16 C for two hors Hold at 4 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 25

28 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Procedre 1 Prepare Second Strand Synthesis Master Mix by adding the following to a 1.7 ml tbe on ice. Pipette 10 times to mix. Second Strand Synthesis Component Second Strand Bffer (SSB) 18 Second Strand Enzyme (SSE) 9 Volme (µl) for 1 Cartridge (2 Samples) 2 Using a P20 single channel pipette, add 12 µl of Second Strand Master Mix to each sample well. 3 Using a P200 mltichannel pipette set to 40 μl, pipette to thoroghly mix each sample well. 4 Seal the plate and centrifge at 280 g for 10 seconds. 5 Place on the preprogrammed thermal cycler and rn the Second Strand Synthesis (SSS) program. SAFE STOPPING POINT If yo are stopping, leave the plate on the thermal cycler at 4 C overnight or store at -25 C to -15 C for p to two days. Clean Up cdna This process ses Prification Beads (SPB) to prify the library DNA and provides a selection step that removes short library fragments. Consmables Resspension Bffer (RSB) Prification Beads (SPB) Freshly prepared 80% ethanol (EtOH) Pipettes P20 single channel and P200 single channel and mltichannel 96-well plate seal Eqipment Magnetic peg stand (Thermo Fisher, catalog # AM10027) DynaMag 96 Side Magnet (Thermo Fisher, catalog # 12331D) or the DynaMag 96 Side Skirted Magnet (Thermo Fisher catalog # 12027) Abot Reagents See Handling Prification (Magnetic) Beads on page 4 for details abot working with Prification Beads (SPB). Abot Magnets This procedre ses two types of magnetic stands. Both are needed in this protocol and are not interchangeable. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 26

29 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Magnet Type Illstration Step Procedre Magnetic Peg Stand Use this magnet with Bind on page 27 and Wash on page 28. DynaMag 96 Side Magnet or DynaMag 96 Side Skirted Magnet Use either DynaMag magnet with Elte on page 28. Gidelines Use a single channel pipette to transfer Prification Beads (SPB) to sample wells. Using a mltichannel pipette reservoir and a mltichannel pipette reslts in inadeqate Prification Beads (SPB) reagent volme needed to complete this protocol. This process reqires both a magnetic peg stand and either DynaMag side magnet. Preparation 1 Prepare the following consmables: Item Storage Instrctions Resspension Bffer (RSB) 2 C to 8 C Do not discard ntil the protocol is complete. Prification Beads (SPB) 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. Procedre Bind 1 Centrifge sample plate at 280 g for 30 seconds. 2 Vortex Prification Beads (SPB) ntil well-dispersed. 3 Use a P200 single channel pipette to add 44 µl Prification Beads (SPB) to each sample well. Pipette mix ntil evenly distribted (10 15 times). 4 Incbate at room temperatre for five mintes. 5 Place on a magnetic peg stand ntil the liqid is clear (~five mintes). Use a magnetic peg stand ntil Elte on page Use a P200 pipette, set to 120 µl, to removeand discard all spernatant from each well. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 27

30 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Wash 1 Wash two times, as follows. a b c Add 200 µl freshly prepared 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 2 Air-dry on the magnetic peg stand for five mintes. 3 Using a P20 pipette, remove residal 80% EtOH from each well. Elte 1 Remove from the magnetic peg stand. 2 Using a P20 single channel pipette, add 11 µl Resspension Bffer (RSB) to each sample well. Pipette to mix, making sre all beads are resspended. CAUTION Yield loss can occr if beads are not thoroghly resspended. 3 Incbate at room temperatre for two mintes. 4 Seal the plate and centrifge at 280 g for 10 seconds to bring entire soltion to the bottom of the well. 5 Place on a DynaMag 96 side magnet and wait ntil the liqid is clear (~two mintes). 6 Transfer 10 µl of spernatant from each sample well to a new sample well of a 96-well plate. Check Libraries Perform the following optional procedre for qality control analysis on yor sample library. 1 Rn 1 µl of ndilted library on an Agilent Technology 2100 Bioanalyzer sing a High Sensitivity DNA chip. 2 Drag the ble regions to captre the bp range. 3 Record the cdna library fragment size and cdna yield. See Lab Tracking Chart on page 39. An example of the reslting cdna prepared sing this protocol is shown in Figre 19. cdna yields are calclated as follows: X ng/µl * 10 l of cdna = ng cdna yield.cdna yields for PBMC samples can be as low as 0.5 ng, with an average of 1.85 ng. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 28

31 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Figre 19 Example of Bioanalyzer cdna Prodct for PBMC Tagment cdna This step ses the Nextera SreCell transposome to tagment cdna, which is a process that simltaneosly fragments and tags DNA with adapter seqences in a single step. Consmables Tagment Bffer (TCB) Tagment Enzyme (TCE) Tagment Stop Bffer (TSB) 96-well plate seal Pipettes P20 single channel and P200 single channel Preparation 1 Prepare the following consmables. Item Storage Volme (µl) Tagment Bffer (TCB) -25 C to -15 C Thaw on ice. Vortex to mix, and then centrifge briefly. Tagment Enzyme (TCE) -25 C to -15 C Thaw on ice. Gently invert the thawed tbes 3 5 times, and then centrifge briefly. Tagment Stop Bffer (TSB) 15 C to 30 C Check for precipitates. If present, vortex ntil all particlates are resspended. 2 Save the following Tagmentation Program (TGM) on the thermal cycler. Choose the preheat lid option (105 degrees) Set the reaction volme to 40 µl 55 C for five mintes Hold at 4 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 29

32 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Procedre 1 Prepare Tagmentation Mix in a 1.7 ml tbe on ice as follows. Pipette 10 times to mix. Tagmentation Mix Component Tagment Bffer (TCB) 44 Tagment Enzyme (TCE) 22 Volme (µl) for 1 Cartridge (2 Samples) 2 Add 30 µl of Tagmentation Mix to each sample well. Mix with pipette. 3 Seal the plate and centrifge at 280 g for 10 seconds. 4 Place on the preprogrammed thermal cycler and rn the TGM program. 5 Remove the plate from the thermal cycler as soon as the temperatre reaches 4 C. Do not leave the PCR plate on the thermal cycler for longer than six mintes. 6 Remove the seal careflly to avoid cross-sample contamination. 7 Use a P20 pipette to add 10 µl of Tagment Stop Bffer to each well. Pipette to mix with a P200 pipette. 8 Seal the plate and centrifge at 280 g for 10 seconds. 9 Incbate at room temperatre for five mintes. Amplify Tagmented cdna This step ses a 15-cycle PCR program to amplify tagmented cdna and add DNA adapters reqired for clster generation. To ensre that yor libraries prodce high-qality seqencing reslts, se the specified nmber of PCR cycles. Consmables Tagmentation PCR Mix (TPM) Tagment PCR Adapter (TPP1) DNA Adapters (N7XX) 96-well plate seal Pipettes P20 single channel and P200 single channel and mltichannel Preparation 1 Prepare the following consmables. Item Storage Instrctions DNA Adapters -25 C to -15 C Only remove adapters being sed. Thaw at room temperatre for 20 mintes. Vortex to mix, and then centrifge briefly. Tagmentation PCR Mix (TPM) -25 C to -15 C Thaw on ice. Gently invert the thawed tbes 3 5 times, and then centrifge briefly. Tagment PCR Adapter (TPP1) -25 C to -15 C Thaw at room temperatre for 20 mintes. Vortex to mix, and then centrifge briefly. 2 Save the following Library Amplification (LA) program on the thermal cycler: Choose the preheat lid option and preheat to 105 C Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 30

33 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Set the reaction volme to 100 µl 95 C for 30 seconds 15 cycles of: 95 C for 10 seconds 60 C for 45 seconds 72 C for 60 seconds 72 C for five mintes Hold at 4 C Procedre 1 Arrange the DNA Adapters in a tbe rack. Use a different index for each sample well. Record the DNA Adapter index sed for each sample well. This information will be reqired when setting p the seqencing rn. 2 Using a P200 single channel pipette, add 30 µl of Tagmentation PCR Mix (TPM) to each of the tagmented samples. 3 Using a P20 single channel pipette, add 10 µl of Tagment PCR Adapter (TPP1) to each of the tagmented samples. 4 Using a P20 single channel pipette, add 10 µl of each DNA Adapter to each tagmented sample. DNA Adapters are one time se and do not reqire new orange caps. 5 Use a P200 mltichannel Pipette to mix times. 6 Seal the plate and centrifge at 280 g at 20 C for 30 seconds. 7 Place on the preprogrammed thermal cycler and rn the LA program. SAFE STOPPING POINT If yo are stopping, leave the plate on the thermal cycler at 4 C overnight or store at -25 C to -15 C for p to two days. Clean Up Libraries This process ses Prification Beads (SPB) to prify the library DNA and provides a selection step that removes short library fragments. Consmables Resspension Bffer (RSB) Prification Beads (SPB) Freshly prepared 80% ethanol (EtOH) Pipettes P20 single channel and P200 single channel and mltichannel 96-well plate seal Eqipment Magnetic peg stand (Thermo Fisher, catalog # AM10027) DynaMag 96 Side Magnet (Thermo Fisher, catalog # 12331D) or the DynaMag 96 Side Skirted Magnet (Thermo Fisher catalog # 12027) Abot Reagents Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 31

34 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) See Handling Prification (Magnetic) Beads on page 4 for details abot working with Prification Beads (SPB). Abot Magnets This procedre ses two types of magnetic stands. Both are needed in this protocol and are not interchangeable. Magnet Type Illstration Step Procedre Magnetic Peg Stand Use this magnet with Bind on page 32 and Wash on page 33. DynaMag 96 Side Magnet or DynaMag 96 Side Skirted Magnet Use either DynaMag magnet with Elte on page 33. Gidelines Use a single channel pipette to transfer Prification Beads (SPB) to sample wells. Using a mltichannel pipette reservoir and a mltichannel pipette reslts in inadeqate Prification Beads (SPB) reagent volme needed to complete this protocol. This process reqires both a magnetic peg stand and either DynaMag side magnet. Preparation 1 Prepare the following consmables. Item Storage Instrctions Resspension Bffer (RSB) 2 C to 8 C Do not discard ntil the protocol is complete. Prification Beads (SPB) 2 C to 8 C Let stand for 30 mintes to bring to room temperatre. 2 Prepare fresh 80% ethanol from absolte ethanol. Procedre Bind 1 Centrifge sample plate at 280 g for 30 seconds. 2 Vortex Prification Beads (SPB) ntil well-dispersed. 3 Using a P200 single channel pipette, add 58 µl of Prification Beads (SPB) to each sample well. Pipette to mix, making sre that all beads are resspended. 4 Incbate at room temperatre for five mintes. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 32

35 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) 5 Place on a 96-well magnetic peg stand ntil the liqid is clear (~five mintes). Use a magnetic peg stand ntil Elte on page Remove and discard all spernatant from each well. Wash 1 Wash two times, as follows. a b c Add 200 µl freshly prepared 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 2 Using a P20 pipette, remove residal 80% EtOH from each well. 3 Air-dry on the magnetic peg stand for five mintes. Elte 1 Remove from the magnetic peg stand. 2 Using a P200 pipette, add51 µl of Resspension Bffer (RSB) to each sample well. Pipette mix ntil beads are thoroghly resspended. 3 Incbate at room temperatre for two mintes. 4 Apply the seal and centrifge at 280 g for 10 seconds to bring entire soltion to the bottom of the well. 5 Place on a DynaMag 96 side magnet ntil the liqid is clear (~two mintes). Use a DynaMag 96 side magnet ntil Second Bind on page Transfer 50 µl of spernatant from each sample well to a new sample 96-well plate. Second Bind 1 Vortex Prification Beads (SPB) ntil well-dispersed. 2 Add 30 µl of Prification Beads (SPB) to each sample well. Use a P200 to pipette ntil evenly distribted (10-15 times). 3 Incbate at room temperatre for five mintes. 4 Place on a magnetic peg stand ntil the liqid is clear (~five mintes). Use a magnetic peg stand ntil Second Elte on page Remove and discard all spernatant from each well. Second Wash 1 Wash two times, as follows. a b c Add 200 µl freshly prepared 80% EtOH to each well. Incbate on the magnetic stand for 30 seconds. Remove and discard all spernatant from each well. 2 Using a P20 pipette, remove residal 80% EtOH from each well. 3 Air-dry on the magnetic peg stand for five mintes. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 33

36 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Second Elte 1 Remove the 96-well plate from the magnetic peg stand. 2 Add 11 µl Resspension Bffer (RSB) to each sample well. Using a P200, pipette mix ntil beads are resspended. 3 Incbate at room temperatre for two mintes. 4 Seal the plate and centrifge at 280 g for 10 seconds to bring entire soltion to the bottom of the well. 5 Place on a DynaMag 96 side magnet ntil the liqid is clear (~two mintes). 6 Transfer 10 µl of spernatant from each sample well to a new 96-well plate. SAFE STOPPING POINT If yo are stopping, seal the plate and store at -25 C to -15 C for p to seven days. Assess Libraries Accrately qantify DNA libraries to ensre optimm clster densities on the flow cell. 1 Rn 1 µl ndilted library on an Agilent Technology 2100 Bioanalyzer sing a High Sensitivity DNA chip. 2 Determine the concentration of the library sing the Agilent Technology 2100 Bioanalyzer. 3 Select the Region Analysis tab. 4 Drag the ble region lines to captre the bp region. Record the final library fragment size and final library yield. See Lab Tracking Chart on page 39. The following figre shows an example trace of a sccessflly seqenced library. Typical libraries show a broad size distribtion ~ bp. A wide variety of libraries can be seqenced with average fragment sizes as small as 450 bp or as large as 1200 bp. Figre 20 yield Example of Library Size Distribtions for PBMC: ble trace = 0.4 nm library yield. Red trace nm library Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 34

37 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) 5 Typical library yield is nm, bt yields as low as 0.5 nm have been observed and sccessflly seqenced on NextSeq. For more information, see the NextSeq System Denatre and Dilte Libraries Gide ( ). For library yields > 2nM, normalize samples to 2 nm. Prepare for Seqencing The PBMC protocol is intended for se only on the NextSeq platform. Use the SreCell Seqencing Primer for yor seqencing rns. The Seqencing Primer (SP) is concentrated at 50 μm and mst be dilted according to the cstom seqencing primer docmentation for the NextSeq platform. See Additional Resorces on page 1. Confirm that Illmina Experiment Manager v1.13 or later is sed to set p the seqencing sample sheet if the BaseSpace Prep Tab is not sed. Version 1.13 or later has the appropriate UMI settings and index seqences for sample demltiplexing. If demltiplexing otside of BaseSpace Seqence Hb, confirm that bcl2fastq v2.18 or later is sed for FASTQ Generation. NOTE Consmables SreCell Seqencing Primer is compatible with this library and PhiX only. Seqencing Primer (SP) (50 μm) [Optional] PhiX Control v3 Cstom Primer Gide NextSeq System Cstom Primers Gide (docment # ) NextSeq Loading Concentration The SreCell loading concentration for the NextSeq platform is 3pM, based on BioAnalyzer qantification. If yo are qantifying with another method, yo may need to optimize the loading concentration. Seqence in Yor Lab 1 Follow the instrctions for sing cstom primers for a seqencing rn on the NextSeq platform. See Additional Resorces on page 1. CAUTION For NextSeq rns connected to BaseSpace Seqence Hb, select the SreCell WTA 3' Library Prep kit dring Prep Tab setp to ensre that the Cstom Primer R1 option is atomatically selected on the Planned Rns screen. This option mst be selected or the seqencing rn fails. NOTE [Optional] Add a 1% PhiX control spike-in as a positive control for alignment and error rate calclations. For more information, see the PhiX Control v3 spport page on the Illmina website. Seqence Using an Otside Lab 1 Conslt with yor seqencing lab abot diltion of Seqencing Primer (SP) and dilte accordingly. 2 Send Seqencing Primer (SP) with the qantified libraries. The lab adds the Seqencing Primer (SP) to the appropriate seqencing reagents for the Illmina instrment sed for seqencing. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 35

38 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) 3 [Optional] Add a 1% PhiX control spike-in as a positive control for alignment and error rate calclations. For more information, see the PhiX Control v3 spport page on the Illmina website. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 36

39 Appendix A Spporting Information Spporting Information Introdction 37 How does the SreCell WTA 3' Assay Work? 37 Cell Conting Protocol 37 Lab Tracking Chart 39 Acronyms 40 Kit Options 40 Consmables and Eqipment 43 Introdction The protocol described in this gide assmes that yo have reviewed the contents of this section, confirmed workflow contents, and obtained all reqired consmables and eqipment. How does the SreCell WTA 3' Assay Work? Single cells are individally partitioned into sbnanoliter droplets on a disposable cartridge sing the Bio-Rad ddseq Single-Cell Isolator. Cell lysis and cell barcoding of mrna transcripts takes place in each droplet dring reverse transcription. Then droplets are disrpted, and the barcoded cdna is pooled for second strand synthesis in blk. Doble-stranded cdna is tagmented by Nextera SreCell transposome to add primer binding sites for sbseqent indexing and amplification by PCR. Final libraries are prified and ready for seqencing on Illmina seqencing platforms. Cell Conting Protocol Follow these steps to perform cell conting on PBMC. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 37

40 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Assess concentration and viability of the PBMC sspension after the washes and final resspension in PBS+0.1% as described in Prepare, Cont, and Assess Viability of Single-Cell Sspension on page 8. 1 Perform cell viability measrement as follows: a Vortex the cell sspension tbe for a few seconds. b Use a P20 pipette to take 10 µl from the middle of the cell sspension, and then add 10 µl of trypan ble 0.4%. c Pipette 10 times to mix, then load onto Chamber A of the Bio-Rad TC20 conting slides. d Measre the cell viability (shold be 80%). NOTE Make sre the cell sspension is stored on ice at all times. 2 Perform cell concentration measrement as follows: a b c d e f g Vortex the cell tbe for a few seconds. Use a P20 pipette to take 10 µl from the middle of the cell sspension, and immediately load it onto Chamber B of the TC20 chip. Measre the cell conts in Chamber B on the TC20. This step shold be performed withot adding trypan ble. Vortex the cell tbe for a few seconds. Use a P20 pipette to take 10 µl from the middle of the cell sspension and immediately load it onto Chamber A of the TC20 chip. Take another 10 µl from the middle of the cell sspension and immediately load it onto Chamber B of the TC20 chip. Measre the cell conts in Chamber A and B on the TC20. This step shold be performed withot adding trypan ble. NOTE Make sre the cell sspension is stored on ice at all times. 3 Calclate the average cell concentration with the three measrements performed in step 2 to arrive at an accrate cell cont. 4 Check the average cell concentration. Dilte or concentrate the cell sspension if cells are above or below the target range of 3000 cells/µl (do not go nder 2500 cells/µl). Use cold PBS + 0.1% BSA to dilte or resspend. 5 Proceed to Prepare Cell and Barcode Sspension Mixes on page 9. Store cell sspension on ice with preparing reagent. Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 38

41 Illmina Bio-Rad SreCell WTA 3' Library Prep for Peripheral Blood Mononclear Cells (PBMC) Lab Tracking Chart Record lab tracking information and sample observations throghot the protocol. The following template can be sed to record process control reslts sch as cdna yield and final library yield. Have this information available when contacting Illmina Technical Spport. In addition to the observation form, record the following information if yo have nexpected reslts. A detailed description of the problem The steps that were performed immediately before the problem occrred The expected reslts The observed reslts Docment # v00 For Research Use Only. Not for se in diagnostic procedres. 39