Single Cell Sample Submission Guideline

Transcription

1 Document NO.: SOP-SMM-016 Version: A1 Effective Date:

2 Document NO:SOP-SMM-016 Version: A1 Page 1 of 8 Content CHAPTERⅠREQUIREMENTS FOR SINGLE CELL OR FEW CELLS ( CELLS ISOLATED BY CUSTOMERS) SINGLE CELL RE-SEQUENCING SAMPLE REQUIREMENT SAMPLE PREPARATION SINGLE CELL WHOLE GENOME BISULFITE SEQUENCING SINGLE CELL TRANSCRIPTOME/ RNA-SEQ (SMART-SEQⅡ) SAMPLE REQUIREMENT SAMPLE PREPARATION SAMPLE PACKAGE AND SHIPMENT CELL SORTING ENVIRONMENT AND OPERATIONAL REQUIREMENTS... 5 CHAPTER Ⅱ SAMPLE REQUIREMENTS FOR CELL LINES, BLOOD AND TISSUE (CELLS ISOLATED BY BGI ) CELL LINES BLOOD CELLS TISSUE SAMPLES ADDITIONAL NOTES... 8

3 Document NO:SOP-SMM-016 Version:A1 Page 2 of 8 ChapterⅠRequirements for Single Cell or few cells ( cells isolated by customers) 1 Single cell re-sequencing 1.1 Sample Requirement This method is mainly for human or mice. It is recommended to provide housekeeping gene detection primers for other species. 1.2 Sample Preparation Cells are sorted into 200μL PCR tube containing 3μL~5μL PBS buffer and should not exceed 1000 cells per tube. It is not recommended to add any other buffer or solution. Please make notes clearly if cells were treated with any special reagent or treatment After completing isolation, the cells should be stored at -80 or dry ice within10 minutes. Or cells can be stored at -20 temporarily for 3h It is recommended to prepare 3 replicates for each sample to ensure the success rate of the follow-up experiments. (For example, if performing sequencing for 5 samples, we propose to send us 15 cells separately.) Additionally, a negative control sample should be provided in each batch, by adding 0.1μL ~0.5μL of 1% PBS-BSA buffer (cell preservation solution when sorting cells). Table1. Sample Requirements for Single Cell DNA-seq Sequencing Type Sample Type WGS WES Single cell 1-2 cells in 4μL PBS buffer 1-2 cells in 4μL PBS buffer Few cells 2 X 1000(4μL PBS buffer) 2 X 1000(4μL PBS buffer) Note:X represents cell number. 2 Single Cell Whole Genome Bisulfite Sequencing 2.1 Sample preparation The lysis buffer for single cell WGBS (Whole Genome Bisulfite Sequencing) is provided by BGI. Please contact our local sales staff μL lysis buffer is stored in 200μL PCR tube and should be kept -80 after receiving.

4 Document NO:SOP-SMM-016 Version:A1 Page 3 of Thaw the lysis buffer on the ice before ready to use Transfer cell(s) into the lysis buffer, and then put the lysis buffer on ice immediately The samples were incubated at 37 C for 1 hour in a PCR machine The lysed cell(s) could be stored at -80 temporarily or transport with dry ice immediately It is recommended to prepare 3 replicates for each sample to improve the success rate of the following experiments. 3 Single Cell Transcriptome/ RNA-seq (SMART-seqⅡ) 3.1 Sample Requirement a) Eukaryote (animal), mrna with poly (A) +. b) Single cell: cell diameter greater than 10μm. Such as oocyte, single cell isolated from morula or cell lines. c) Few cells: cell number is less than 200, such as morula, blastocyst. d) Total RNA of picogram or more: 28S/18S 1,RIN 7,concentration>50pg/μL. Table 2 Sample Requirements for Single Cell RNA-seq Sequencing type Sample type Transcriptome RNA-Seq( Quantification) Single cell 1-2 cells(4μl lysis buffer) 1-2cells(4μL lysis buffer) Few cells 2 X 200(4μL lysis buffer) 2 X 200(4μL lysis buffer) Total RNA Total RNA>2ng ; RNA 28S/18S 1, RIN 7, concentration>50pg/μl Total RNA>2ng ; RNA 28S/18S 1,RIN 7, concentration>50pg/μl Note:X represents cell number

5 Document NO:SOP-SMM-016 Version:A1 Page 4 of 8 Table3 Sample Classification for Single Cell RNA sequencing Sample Classification Cell Type Risk Evaluation Level A Oocyte, blastomere, early embryo Success rate is greater than 90%. Level B Cells isolate from embryo, cell line, tissue(cell diameter>10μm) Low Risk Level C Complex procedure or special treatment,e.g. CTC High Risk Level D Special cell Infeasible Additional information: the success rate of single cell amplification is affected by cell type, cell sorting method, cell sorting operation skills, transport condition, etc. The risk classification is for reference only and it subjects to the actual experiment. Special cells, for example, cells that carry large part of fat, protein, polysaccharide or other components, or plant cells which contain cell wall, may affect the enzyme reaction, thus influence the amplification effect. It is recommended to provide test samples for pre-experiment. 3.2 Sample Preparation The lysis buffer for single cell SMART-seqⅡis provided by BGI. Please contact our local sales staff μL lysis buffer is stored in 200μL PCR tube and should be kept -80 after receiving Thaw the lysis buffer on the ice before ready to use Collecting cells: Cell isolation should be done as little as 30 minutes. Transfer cell(s) into lysis buffer. The volume of single-cell droplet added to the lysis buffer should be no more than 0.3μL, and the final volume of droplets containing cells should be no more than 1μL. And then put the tube on ice Centrifuge the PCR tube at 4 gently and store at -80 or dry ice within10 minutes.

6 Document NO:SOP-SMM-016 Version:A1 Page 5 of It is recommended to prepare 3 replicates for each sample to ensure the success rate of the following experiments. Additionally, a negative control sample should be provided in each batch, by adding 0.1μL~0.5μL of 1% PBS-BSA solution (cell preservation solution when sorting cells). 4 Sample Package and Shipment 4.1 Fill out the samples information sheet and send along with the samples. In addition to the samples basic information, it also includes: a) Cell type b) Cell sorting method and date c) Cell number in each PCR tube d) Please fill in the following information as far as possible: Content of nucleic acid in the cell; physicochemical characteristic and morphological structure of the cell, such as cell size and inclusion 4.2 Label the sample on PCR tubes, and put the PCR tubes into sealed bags. Then send the samples to BGI with adequate dry ice as soon as possible. From cells isolation to reach BGI, the whole time of this process should not exceed one week. Please add enough dry ice to avoid freezing and thawing of the cell sample during shipment. 5 Cell sorting environment and operational requirements Cells isolation should be performed in a clean space (eg cell culture lab.). Please wear disposable gloves and masks, and clean the gloves and work bench with 70% ethyl alcohol. It is recommended to use RNA-zap (Ambion ) to wipe the work bench and related equipment before the transcriptome/ RNA seq library construction. Do not touch other instruments that are irrelative to the experiment during operation. The consumables and reagents used in the experiment should be Nuclease-Free.

7 Document NO:SOP-SMM-016 Version:A1 Page 6 of 8 Chapter Ⅱ Sample Requirements for cell lines, blood and tissue (cells isolated by BGI ) Customers can also send us processed cell suspension for cell isolation. BGI use mouth pipette to randomly isolate single cell. The sample is treated as follows: 1 Cell lines a) Make sure the cells are in good condition when performing microscope examination, and the cell density is not less than 80%. b) Resuspend cells with cell freezing medium, to a final cell density of /ml, and then dispense the cells suspension to the cryopreserved tubes, the total volume of each tube is no less than 500μL. (Cell cryoprotective agents: 90% fetal bovine serum (FBS) + 10% DMSO). c) Freezing method: it is recommended to use gradient freezing container. Transfer 250mL of fresh isopropyl alcohol (it can be re-used for 3-5 times) to gradient freezing container at room temperature. Place the cryopreservation tube into the gradient freezing container, 4 for 30 min, -20 for 30-60min, and then -80 overnight. After being removed from the freezing container, tubes contains cells can be kept at -80 for short-term storage, or keep them in liquid nitrogen for long-term preservation. Transport samples with enough dry ice. 2 Blood cells All operations were performed under aseptic conditions: a) Draw 5mL human peripheral blood into an EDTA vacuum blood collection tube. Incubate lymphocyte separation medium (LSM) and blood in 20 water bath for 20 minutes separately. b) Dilute the blood with equal volume of HBSS solution. c) Transfer 2mL LSM into a 15mL centrifuge tube, keep the tube at an angle of 45, pipet diluted blood and put the pipette 1cm away from the stratified liquid surface, then add the blood into the centrifuge tube along tube wall slowly, to maintain a clear distinct blood-lsm interface. (The volume ratio of the blood sample to the stratified liquid is about 2: 1).

8 Document NO:SOP-SMM-016 Version:A1 Page 7 of 8 d) Centrifuge the tube at 500 g for 20min at 20 (Centrifuge must be set to no break in the deceleration settings). After centrifugation, the contents of the tube is divided into three layers, the upper layer of plasma (containing cell debris), the middle layer of the stratified liquid, the bottom of red blood cells. In the upper and middle layer of liquid interface can be seen in milky white turbid mononuclear cell layer (buffy coat), mononuclear cells including lymphocytes and monocytes. e) Discard the upper plasma, carefully aspirate mononuclear cells and lymphocytes and transfer them to a new 15mL centrifuge tube. f) Add 5mL HBSS to the centrifuge tube and centrifuge at 300 g for 10min, then discard the supernatant. g) Resuspend cells in 1mL HBSS and count them under microscope, then freeze the cells at the final concentration of /ml (Cell freezing medium: 90%FBS+10%DMSO). h) Cryopreservation and transport are consistent with cell lines preparation method. 3 Tissue samples Tissue samples would be digested into cells suspension before cryopreservation. a) After surgery, fresh tissue would be immersed into a tube which contains PBS/HBSS and then transferred to a clean bench. Tissue would be washed once time with PBS/HBSS and then cut into 0.5 mm 3 pieces in 60mm dish with a sterile scissors immediately. b) Take the epithelial tissue for instance, digest tissue into cells suspension with the following method : incubate at 37 for 2 hours, and mix up at 30-minute intervals. Reagent Volume/concentration Tissue homogenate Medium(90%DMEM+10%FBS) Collagenase I(Gibco Cat.No ) Collagenase IV(Gibco Cat.No ) X (5-10) * X 1mg/mL 0.5mg/mL Note: For different tissue types using different enzymes and digestive methods according to the literature or commercial kit instructions.

9 Document NO:SOP-SMM-016 Version:A1 Page 8 of 8 c) After digestion, cells would be filtered by passing them through a sterile 40µm cell strainer. Centrifuge filtrate at 500 g for 5 minutes at 4. Discard the supernatant and keep the precipitation in tube bottom. d) Cell pellet would be resuspended with 1mL HBSS and counted under microscope, and then would be freezed at a final concentration of /mL (Cell freezing medium, 90%FBS+10%DMSO). e) Cryopreservation and transport are consistent with cell lines preparation method. 4 Additional notes Single cell samples could be prepared according to your own protocols. However, cryopreservation method (cell density, freezing medium, frozen storage temperature, etc.) should be operated strictly according to the protocol.