title: GSM5511355: S3_T0; Homo sapiens; RNA-Seq
library_construction_protocol: Human peripheral blood was collected from healthy donors and septic patients. Blood was recovered in Vacutainer glass collection tubes with heparin (BD Biosciences). Peripheral blood mononuclear cells (PBMC) were isolated by gradient centrifugation with Histopaque-1077(400g,30min at room temperature). PBMC cells at the interface of plasma and Histopaque were harvested by paster pipette then washed twice with growth medium (RPMI 1640, 10% FBS, 100 U/ml penicillin, 100 ug/ml streptomycin, 2 mM L-glutamine). Cell aliquots were frozen in liquid nitrogen with 10%DMSO and 90% FBS. For single cell sequencing, cryovials were thawed in the water bath at 37°C for 2-3 min. In a biosafety hood, thawed cells were transferred to a 50 ml conical tube using a wide-bore pipette tip. Cryovial was rinsed with 10 ml warm complete growth medium. Cell suspension was centrifuged at 300g for 5 min. PBMC live cells were recovered by dead cell removal kit (Miltenyi) and viable cells confirmed by hemocytometer counting (>85% viable). 2 x 106 cells were transferred into a new 50-ml tube and centrifuged at 300g for 5 min. Supernatant was gently removed without disrupting the cell pellet, which was then re-suspended in 1 ml 1X PBS + 0.04% BSA and gently mixed by pipette. Cells were transferred into a 2ml microcentrifuge tube then centrifuged, 300g for 5 min, then supernatant removed without disrupting the cell pellet. Cells were resuspended in an appropriate volume of 1X PBS + 0.04% BSA to obtain a concentration of 700-1,200 cells/μl then placed on ice. 15,000 cells/sample single-cell suspensions were loaded into a Chromium Single Cell Chip (10x Genomics) according to the manufacturer's instructions. The single-cell RNA-seq libraries for Illumina sequencing using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x Genomics) was performed according to the manufacturer's instructions. Samples were sequenced at UCSD Genomics center on the NovaSeq platform at 250M reads/sample.