title: GSM7207794: PBMC, R-P4, myocarditis, recovered, BCR; Homo sapiens; RNA-Seq
library_name: GSM7207794
library_construction_protocol: PBMCs were first isolated by Ficoll-Paque PLUS (GE Healthcare) or Lymphoprep (STEMCELL Technologies) density gradient centrifugation. Cells were subsequently washed twice in phosphate buffered saline (PBS) and resuspended in complete RPMI 1640 (cRPMI) medium (Lonza) containing 10% fetal bovine serum (FBS), 2 mM glutamine, and 100 U/ml each of penicillin and streptomycin (Invitrogen). PBMCs were then stored at 10^6 cells/ml in 10% dimethyl sulfoxide (DMSO) in FBS and stored in -80 °C overnight before further storage in liquid nitrogen. Serum was isolated by centrifugation of serum tubes and saving the supernatant in aliquots, which were flash frozen in liquid nitrogen prior to cryopreservation in -80 °C. All patient PBMCs were processed and cryopreserved within 1-6 hours of blood draw. Cryopreserved PBMCs were thawed in a water bath at 37 °C for ~2 minutes and removed from the water bath when a tiny ice crystal remained. Cells were transferred to a 15 mL conical tube of pre-warmed growth medium. The cryovial was rinsed with additional growth medium (10% FBS in DMEM) to recover leftover cells, and the rinse medium was added to the 15 mL conical tube while gently shaking the tube. Thawed PBMCs were centrifuged at 400g for 8 minutes at RT, and the supernatant was removed without disrupting the cell pellet. The pellet was resuspended in 1X PBS with 2% FBS, and cells were filtered with a 30 μM cell strainer. Cellular concentration was adjusted to 1,000 cells/μL based on the cell count and cells were immediately loaded onto the 10x Chromium Next GEM Chip G, according to the manufacturer's user guide (Chromium Next GEM SingleCell V(D)J Reagent Kits v1.1). We aimed to obtain a yield of ~10,000 cells per lane. cDNA libraries for gene expression and TCR/BCR sequencing were generated according to manufacturer's instructions (Chromium Next GEM SingleCell V(D)J Reagent Kits v1.1). Each library was then sequenced on an Illumina NovaSeq 6000 platform.