GSM7413340: C148 B cell, elderly, replicate 1, VDJ (BCR); Homo sapiens; RNA-Seq
Venous blood samples were collected into Heparin and Serum-gel monovette tubes by standard phlebotomy at The Rockefeller University. Peripheral Blood Mononuclear Cells (PBMCs) obtained from samples collected were further purified as previously reported by gradient centrifugation and stored in liquid nitrogen in the presence of Fetal Calf Serum (FCS) and Dimethylsulfoxide (DMSO). Peripheral blood mononuclear cells (PBMC) were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer's instructions. Library was performed according to the manufacter's instructions (single cell 5' v2 protocol, 10x Genomics). Briefly, B cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropriated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences were added. V(D)J libraries were generated with 350 the Chromium Single Cell 5′ Library & Gel Bead Kit and 351 Chromium Single Cell V(D)J Enrichment Kit, Human B Cell (10x Genomics protocol)