design: scRNASeq of Naïve, GC, plasma, memory B cells sorted from WT and Fip200-deficient mice spleen
library_construction_protocol: FIP200f/fMb1Cre+/- (KO) mice and their littermatecontrol, FIP200f/fMb1Cre-/-(WT) were immunized with 50 ug NP29-KLH with Alum, then splenocytes isolated from WT and KO mice 11 days after immunization were enriched for GC, plasma, and memory B cells by depleting non-B cells and IgDhi B cells using magnetic beads. Those enriched samples were then barcoded and sorted for naïve, GC, plasma, and memory B cells, which were then mixed for single-cell RNA sequencing. Mixed 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). Library was performed according to the manufactor's instruction (Chromium Single-Cell 5′ Reagent Kit v2 Chemistry, Dual Index). Briefly, the poly-A RNA form the cell lysate contained in every signle GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, the 10x Barcode and Unique Molecular Identifier (UMI). The pooled barcodeed cDNA was then cleaned up with Sliane DynaBeads, amplified by PCR and the apropiated sized fragmnets were selected with SPRIselect reagent for subsequent library constrution. During the library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and Sample index i5 and i7 were added.