GSM9158862: PBMCs, GBS and control cells, multiplexed; Homo sapiens; RNA-Seq
Frozen cryovials containing 107 PBMCs/mL were thawed in a 37°C water bath for 2 minutes. Following thawing, 1 mL of pre-warmed RPMI 1640 medium (Gibco, 11875-093) supplemented with 10% HyClone™ Fetal Bovine Serum (Thermo Fisher Scientific, SH3008803IR) and 1% penicillin-streptomycin (Thermo Fisher Scientific, 15070063) was added to each vial. The cell suspension was transferred to a 15 mL conical tube, and the cryovials were rinsed with additional RPMI media to maximize PBMC recovery. To aid in cell dissociation, Accumax (Thermo Fisher Scientific, 00-4666-56) was added at a 1:1 ratio to the sample volume, and cells were incubated at room temperature for 5 minutes. Samples were centrifuged at 350g for 5 minutes at room temperature. The supernatant was aspirated, and the cell pellets were resuspended in 1 mL of RPMI media containing 100 µg/mL DNase I and incubated at 37°C for 15 minutes. Cells were washed once with 1X PBS and centrifuged at 500g for 5 minutes. The resulting cell pellets were fixed at 4 °C for 17 hours using the Chromium Single Cell Fixed RNA Sample Preparation Kit (10x Genomics). Fixed cells were subsequently hybridized with probes using the Fixed RNA Feature Barcode Kit (10x Genomics), following the manufacturer's protocol. Remaining library preparation steps were carried out according to the standard 10x Genomics workflow.