GSM7866761: PBMC_scRNA-seq; Homo sapiens; RNA-Seq
PBMCs were isolated from whole blood within 2 hours of collection. The protocol is as follows.  1.Make up media with Dulbeccomicros phosphate-buffered saline (PBS) and 2mM EDTA. 2.Fill a Leucoseps tube with 15 mL Ficoll Paque Plus (room temperature) 3.Centrifuge at 1000 g for 30 seconds. 4.Transfer whole blood from EDTA tubes into a 50 mL Falcon tube. 5.Add equivalent volume of media to the whole blood. 6.Transfer a maximum of 30 mL of blood/media mix into each Ficoll-loaded Leucosep tube. 7.Centrifuge at 800g, 15 minutes, room temperature with deceleration set to 0. 8.Remove the majority of plasma from the Leucoseps tube using a pipette, leaving approximately a 1cm layer of plasma above the cell layer. 9.Gently swirl the Leucoseps tube to mix the remaining contents, and transfer into a new Falcon tube. 10.Top up to 40 mL with media. 11.Centrifuge at 200g at room temperature, ensuring deceleration is set back to 9. 12.When completed, set the centrifuge to 4°C. 13.Gently remove the supernatant using a pipette, resuspend cell pellet using a 100 microL pipette and add 5 mL of red blood cell lysis solution (1 part 10X RBC lysis buffer, 9 parts sterile water). Gently mix and leave at room temperature for 5 minutes. 14.If currently have multiple tubes of PBMCs, these can be combined at this point. 15.Top up to 40mL with media. 16.Centrifuge at 500g, 4°C for 10 minutes. 17.Gently discard supernatant using a pipette. 18.Discard the supernatant and resuspend the pellet using a 100 microL pipette. 19.Bring up the volume to 30-40 mL using media. 20.Count cells. 21.Centrifuge at 500g, 4°C for 10 minutes. 22.Discard supernatant using pipette. 23.Resuspend pellet in residual volume using 100 microL pipette and put cells on ice. 24.Add enough cold Recovery Cell Culture Freezing Medium (Thermo Fisher, 12648010) dropwise to store cells at desired concentration, such as 3 or 6 million cells per mL. 25.Add 1mL of cell suspension to separate cryovials. 26.Place cryovials into a Mr Frosty and transfer to a -80°C freezer. 27.After 24 hours, transfer PBMCs to a -150°C freezer.  Protocol for PBMC thawing and pooling prior to scRNA-seq 1.Prepare medium with RPMI and 10% FBS. Warm to 37°C in a water bath. 2.For each cryovial which will be thawed, label a 15 mL Falcon tube. 3.Thaw cryovial in water bath, without submerging fully, for 2-3 minutes until there is a small ice ball. 4.Wipe cap with 70% ethanol and pour gently into the 15 mL Falcon tube. 5.Add 1 mL media to each empty cryovial (to collect remaining cells), and pipette dropwise into Falcon tube. 6.Add 8mL warmed media dropwise to Falcon tube. 7.Swirl tube gently and centrifuge at 300g for 5 minutes, room temperature. 8.Discard supernatant using a pipette. 9.Resuspend with 100 microL pipette and top up to 1ml with media 10.Count cells. 11.Add 9mL media, and transfer equal number of cells (e.g. 1 million) from each donor into a new 15 mL Falcon tube. 12.Centrifuge at 300g for 5 minutes, room temperature. 13.Discard supernatant and resuspend pellet in residual volume. 14.Add enough media to achieve a concentration of 1500 cells per microL. 15.Pass through a 40 microm filter and repeat count. 16.Transfer 1 mL into a new tube and place on ice until cDNA library preparation. Gene expression libraries were prepared from single cells using the Chromium Controller and Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics, Inc. Pleasanton, USA) according to the manufacturer's protocol (CG000315 Rev E). Briefly, nanoliter-scale Gel Beads-in-emulsion (GEMs) were generated by combining barcoded Gel Beads, a master mix containing cells, and partitioning oil onto a Chromium chip. Cells were delivered at a limiting dilution, such that the majority (90-99%) of generated GEMs contain no cell, while the remainder largely contain a single cell. The Gel Beads were then dissolved, primers released, and any co-partitioned cells lysed. Primers containing an Illumina TruSeq Read 1 sequencing primer, a 16-nucleotide 10x Barcode, a 10-nucleotide unique molecular identifier (UMI) and a 30-nucleotide poly(dT) sequence were then mixed with the cell lysate and a master mix containing reverse transcription (RT) reagents. Incubation of the GEMs then yielded barcoded cDNA from poly-adenylated mRNA. Following incubation, GEMs were broken and pooled fractions recovered. First-strand cDNA was then purified from the post GEM-RT reaction mixture using silane magnetic beads and amplified via PCR to generate sufficient mass for library construction. Enzymatic fragmentation and size selection were then used to optimize the cDNA amplicon size. Illumina P5 & P7 sequences, a sample index, and TruSeq Read 2 sequence were added via end repair, A-tailing, adaptor ligation, and PCR to yield final Illumina-compatible sequencing libraries.