title: GSM9056724: PBMC, Covid Vaccine, Non-responder 4; Homo sapiens; RNA-Seq
library_construction_protocol: Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coat preparations by density gradient centrifugation using Ficoll-Hypaque (GE Healthcare). Cells were washed with DPBS and cryopreserved in 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) in liquid nitrogen. Prior to library preparation, PBMCs were thawed, washed, and resuspended in DPBS with 0.04% bovine serum albumin (BSA). Cell viability and concentration were assessed using trypan blue and Countess II FL (Thermo Fisher). Approximately 10,000 viable cells per sample were loaded onto a 10x Genomics Chromium Single Cell 3' Chip (v3.1 chemistry) for gel bead-in-emulsion (GEM) generation and cDNA synthesis. cDNA was amplified by PCR (11 cycles), and sequencing libraries were prepared following the manufacturer's protocol. Single-cell RNA-seq libraries were prepared according to the manufacturer's protocol (10x Genomics, Chromium Single Cell 3' v3.1). Briefly, approximately 10,000 viable PBMCs per sample were resuspended in the master mix and loaded into a Chromium Single Cell Chip alongside partitioning oil and gel beads to generate gel bead-in-emulsion (GEMs). Polyadenylated RNA from each cell was reverse-transcribed into barcoded cDNA within individual GEMs. Each cDNA molecule incorporated an Illumina R1 primer sequence, a 10x barcode, and a unique molecular identifier (UMI). After reverse transcription, emulsions were broken and the cDNA was purified with Silane DynaBeads. cDNA was then amplified by PCR (11 cycles), and sequencing libraries were prepared using SPRIselect reagent for size selection. During library construction, Illumina R2 primer sequence, P5 and P7 adapters, and a sample index were added to enable paired-end sequencing.