GSM8037785: Donor 4B Burn Whole Blood; Homo sapiens; RNA-Seq
For lymphocyte isolation, whole blood heparinized vacutubes were used. To isolate lymphocytes by immunomagnetic negative selection, 100 μl of Isolation Cocktail and 100 μl of Rapid Spheres (EasySep Direct Human Total Lymphocytes Isolation Kit, 19655, STEMCELL Technologies) were added to 2 ml of whole blood. After mixing and 5-min incubation at room temperature, the sample volumes were topped up to 2.5 ml with 0.04% BSA in PBS. The diluted sample was incubated in the magnet without a lid for 5 min at room temperature, and negatively selected lymphocytes were decanted into a new 5-ml polystyrene tube. Except for the addition of Isolation Cocktail, all steps were repeated once. The final lymphocyte cell suspension was transferred to a 15-ml polypropylene tube, and a volume of 5 ml of 0.04% BSA in PBS was added to the sample. Lymphocytes were precipitated by centrifugation for 5 min at 300g; the supernatant was discarded; and cells were resuspended in 5 ml of 0.04% BSA in PBS. This step was repeated, and cells were resuspended in 100 μl of PBS + 0.04% BSA. Cell density was quantified with a hemacytometer; cell viability was assessed with trypan blue staining (T8154, Sigma-Aldrich); and 7,500 live lymphocytes were transferred to a sterile 1.5-ml microcentrifuge tube.  For leukocyte isolation, 1 ml of whole blood from heparin-containing vacutubes was transferred to 5-ml polystyrene round-bottom tubes, and 12 μl of 0.5 M EDTA was added. Next, 2% FBS in PBS (1 ml) and 50 μl of EasySep RBC Depletion spheres (EasySep RBC Depletion Reagent, 18170, STEMCELL Technologies) were added to immunomagnetically deplete red blood cells. After 5 min of magnet incubation at room temperature, cell suspension containing leukocytes was decanted into a new 5-ml polystyrene tube. To ensure complete removal of red blood cells, red blood cell depletion was repeated, and cell suspension containing leukocytes was decanted into a new 15-ml polypropylene tube. Leukocytes were precipitated by centrifugation at 300g for 5 min at 20 °C and resuspended in 5 ml of 0.04% BSA in PBS. This step was repeated, and leukocytes were resuspended in 2 ml of 0.04% BSA in PBS. Cell viability and cell density were assessed, and 7,500 live leukocytes were transferred to the microcentrifuge tube containing the lymphocyte cell suspension in a total volume of 50 μl of 0.04% BSA in PBS. The sample was prepared according to 10X Genomics ChromiumTM Single Cell 3' Reagent Guidelines v3 Chemistry. Briefly, single cells were sorted into 0.1% BSA–HBSS and partitioned into Gel Bead-In-EMulsions (GEMs) using 10xTM GemCodeTM Technology. This process lysed cells and enabled barcoded reverse transcription of RNA, generating full-length cDNA from poly-adenylated mRNA. DynaBeads® MyOneTM Silane magnetic beads were used to remove leftover biochemical reagents, then cDNA was amplified by PCR over 10 cycles. Quality control size gating was used to select cDNA amplicon size prior to library construction. Read 1 primer sequences were added to cDNA during GEM incubation. P5 primers, P7 primers, i7 sample index, and Read 2 primer sequences were added during library construction. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed using Illumina NovaSeq S2 flow cell at the Center for Health Genomics and Informatics at the University of Calgary.