GSM6288340: B cells from healthy controls 3 and 4, mRNA-derived cDNA; Homo sapiens; RNA-Seq
GSM6288340
Frozen PBMCs were thawed, and B cell population was enriched using magnetic separation (Miltenyi 130-101-638). Samples were next labelled with oligonucleotide-tagged antibodies against CD11c, CD21, CD27 and CD85j (TotalSeq, BioLegend) and hashtagged with four different anti-human CD298/b2-microglobulin/ TotalSeq Hashtag antibodies. After labelled, cell counts were adjusted to 1,035 cells per microlitre before being loaded onto a Chromium Next GEM chip K (10X Genomics, 2000182). Libraries' construction was performed according to Single cell 5' v2 protocol (CG000330 RevA, 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 full-length cDNA from poly-adenylated mRNA and DNA from protein Feature barcode are amplified and separated by size selection fro generating  5' Gene expression libraries, and cell surface protein libraries, respectively. cDNA is  then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated 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 and a sample index were added. The library was quality controlled by Agilent 4150 TapeStation and quantified by RT-PCR using the KAPA library quantification kit for Illumina platforms (Roche, KK4824). Samples were sequenced on an Illumina NovaSeq with a sequencing depth of at least 50, 000 reads per cell.