A cost effective 5 selective single cell transcriptome profiling approach.

Transcription

1 Revision Description Authors Institution Lab Special Instructions Integrated Barcoding A A cost effective 5 selective single cell transcriptome profiling approach. Arguel et al. CNRS - University of Cote d'azur Pascal Barbry - UCA Genomix - IPMC For library preparation after C, cdna were pooled and used with an adaptated tagmentation protocol from Picelli et al. Refer to the documents "Off chip protocol for Ion Torrent sequencing" and "Off chip protocol for Illumina sequencing" for instructions on sample processing and library preparation. These files are included with the script hub download. Be sure to use the appropriate protocol for the sequencing platform you intend to use. Script Summary - Prime Runtime Estimates Barcode 86x (5-0 um diameter cells) 862x (0-7 um diameter cells) 863x (7-25 um diameter cells) Estimate 0 hours, minutes 0 hours, 3 minutes 0 hours, 2 minutes Script Summary - Cell Load and Stain Runtime Estimates Barcode 86x (5-0 um diameter cells) 862x (0-7 um diameter cells) 863x (7-25 um diameter cells) Estimate 0 hours, 2 minutes 0 hours, 50 minutes 0 hours, 4 minutes Script Summary - Sample Prep Runtime Estimates Barcode 86x (5-0 um diameter cells) 862x (0-7 um diameter cells) 863x (7-25 um diameter cells) Estimate 6 hours, 57 minutes 6 hours, 57 minutes 6 hours, 57 minutes Incubation Profile Script Step Operation Temperature (C) Duration (s) Lysis Incubation S Lysis Cooling Manual 0 80 /9

2 RT Incubation RT and Template Switching RT Inactivation RT inactivation PCR adaptor Denature Hot Start PCR adaptor PCR Cycle (3 Step) x8 Denaturation Annealling 64 5 Extension PCR adaptor End Inactivation PCR adaptor Hold Incubation /9

3 Script Reagent Details - Prime Reagent Loading Volume (µl) Notes 200 µl A 200 µl A2 Blocking Reagent 5 µl C Blocking Reagent 5 µl C2 20 µl P 20 µl P2 Reagent Mix Recipe - Prime Blocking Reagent C Blocking RGT (X) C Harvest RGT (X) 3/9

4 Script Reagent Details - Cell Load and Stain Inlet Reuse Cell Mix Flowthrough Outlet Reagent Loading C C2 Instructions Aspirate inlet prior to loading reagents Aspirate inlet prior to loading reagents (862x, 863x only) Volume (µl) Notes LIVE/DEAD staining solution Cell Wash Buffer 7 5 Cell Mix 6 C Reagent Mix Recipe - Cell Load and Stain C Preloading RGT (X) Cell Mix Suspension RGT (2.5X) 40 Cells 250 / μl Total Prep Volume LIVE/DEAD staining solution Cell Wash BUF (X) Calcein AM (4 mm) Ethidium homodimer- (2 mm) /9

5 Total Prep Volume Protocol Worksheet Cell Wash Buffer Cell Wash BUF (X) 5/9

6 Script Reagent Details - Sample Prep Inlet Reuse Flowthrough Outlet Reagent Loading 2 C2 Instructions Aspirate inlet prior to loading reagents Aspirate inlet prior to loading reagents (862x, 863x only) Volume (µl) Notes 24 2 Lysis Buffer 7 3 RT Mix µl each Harvest Inlets PCR reaction Mix 5 each Harvest Outlets Reagent Mix Recipe - Sample Prep C Preloading RGT (X) Special Instructions: Refer to the documents "Off chip protocol for Ion Torrent sequencing" and "Off chip protocol for Illumina sequencing" for instructions on sample harvesting and cleanup. C Harvest RGT (X) 6/9

7 Lysis Buffer Protocol Worksheet Special Instructions: ERCC aliquots are conserved at -80 C. When needed, thaw on ice, make serial dilutions and do not reuse dilutions. For the reverse transcription primer sequence, refer to the files "Oligo Fequences for Ion Torrent Sequencing" or "Oligo Sequences for Illumina Sequencing" included with the script hub download. Tween 20 (0.4%) RNasin Plus RNase Inhibitor (40 U/µl) Set of datp, dctp, dgtp, dttp (20 mm) ERCC RNA Spike IN Mix ( µg/ml) reverse transcritption primer (0 µm) C Loading RGT (20X) PCR Water RT Mix 9 Total Prep Volume Special Instructions: MgCl2 at 25mM is added to complete MgCL2 from first-strand buffer For template switching oligonucleotide sequence, refer to the documents "Oligo Sequences for Ion Torrent Sequencing" or "Oligo Sequences for Illumina Sequencing" included with the script hub download. C Loading RGT (20X) first-strand buffer (5X) DTT (00 mm) Betaïne (5M) RNasin Plus RNase Inhibitor (40 U/µl) MgCl2 (25 mm) SuperScript II reverse transcriptase (200 U/µl) template switching oligonucleotide (50 µm) PCR Water Total Prep Volume 7/9

8 PCR reaction Mix Protocol Worksheet Special Instructions: For IFC prepare a PreMix of 20X reactions without barcode primer. Dispatch 6.53ul in a 96 plate on ice with corresponding barcode primer before loading IFC wells. For primer sequence, refer to the files "Oligo Fequences for Ion Torrent Sequencing" and "Oligo Sequences for Illumina Sequencing" included with the script hub download. Note that the barcode primers used for Ion Torrent and Illumina sequencers are different. C Loading RGT (20X) Kapa HiFi Hot Start ReadyMix PCR Kit (2X) barcode primer (0.25 µm) biotynylated PCR primer (0 µm) optional illumina reverse PCR primer (0 µm) Total Prep Volume 8/9

9 Protocol Reagent Shopping List Reagent Vendor Part Number Kit Part Number Stock Concentration Calcein AM Life Technologies L mm Ethidium homodimer- Life Technologies L mm Tween 20 Sigma P946-50ML 0.4% RNasin Plus RNase Inhibitor Promega N26 40 U/µl Set of datp, dctp, dgtp, dttp Promega U mm ERCC RNA Spike IN Mix ThermoFisher µg/ml reverse transcritption primer 0 µm first-strand buffer ThermoFisher X DTT ThermoFisher mm Betaïne Sigma B0300-VL 5M MgCl2 Sigma M028-0XML 25 mm SuperScript II reverse transcriptase ThermoFisher U/µl template switching oligonucleotide 50 µm Kapa HiFi Hot Start ReadyMix PCR Kit Clinisciences KK2602 2X barcode primer 0.25 µm biotynylated PCR primer 0 µm optional illumina reverse PCR primer 0 µm Fluidigm Reagent Kits Reagent Part Number Stock Concentration PN PN PN PN PN C Blocking RGT C Harvest RGT C Preloading RGT Suspension RGT Cell Wash BUF C Loading RGT PCR Water X X X 2.5X X 20X 9/9