GSM9449238: Patient 15 - post treatment; Homo sapiens; RNA-Seq
Cryopreserved peripheral blood mononuclear cells (PBMC) from trial participants were stored long-term in liquid phase LN2. For sample preparation, cryotubes were partially submerged in a 37°C water bath until mostly thawed, then transferred to 15mL conical tubes with 9mL of pre-warmed, filter-sterilized RPMI Media (Gibco Cat# 11875093) and 10% by-volume fetal bovine serum (FBS; Hyclone Cat# SH30071.01HI). Cells were centrifuged for 5 minutes at 400g at room temperature, decanted, then resuspended in 1mL chilled (4°C) phosphate-buffered saline (PBS; Gibco Cat# 14190144) + 0.04% bovine serum albumin (BSA; Gibco Cat# 15260037) with 0.2U/µl of RNAse inhibitor (Sigma Cat# 3335399001). Initial cell counting and viability were performed using a Countess II automated cell counter (Invitrogen Cat# AMQAX1000) with a 1:1 dilution of trypan blue (0.4% solution, Gibco Cat# 15250061) to confirm sufficient cell yields and greater than 80% viability. Samples were then processed using the following 10X Genomics protocols. Single cells were captured and barcoded using the 10X Chromium controller using its 3' or 10x Flex (probe-based) whole transcriptome kits. Gel Bead-In EMulsions (GEMs) were prepared, followed by cDNA amplification and size-based cleanup. Final libraries were sent for sequencing using NovaSeq and NovaSeqX platforms to target at least 20k reads per cell.