Illumina HiSeq 2500 sequencing; Single-cell RNAseq of phenotypically defined human B cell subsets
PBMCs isolated from a healthy male 25 year old using lithium herapin vacutainers and Ficoll-paque plus centrifugation. Cells were stained in BD Horizon Brilliant stain buffer with (Biolegend: CD19 HIB19 BV421, CD27 M-T271 FITC, IgD AI6-2 BV785, CD10 HI10a BV605, Live/Dead Zombie NIR), doublet and dead cells removed and lymphocytes gated on FSC/SSC, and sorted into Transitional (CD19+IgD+CD27-CD10+), Naïve (CD19+IgD+CD27-CD10-), IgM Memory (CD19+IgD+CD27+), Classical Memory (CD19+IgD-CD27+) and Double Negative (CD19+IgD-CD27-) populations on a FACSAria (BD Biosciences) at 4°C.  10X genomics 5' chemistry according to manufacturer's protocol at 1 FACS sorted population per lane.  10X genomics Illumina prep according to manufacturer's protocol.