Single-cell RNA sequencing of mouse primary B cells to investigate subpopulation markers
Single-cell RNA sequencing of mouse primary B cells to investigate subpopulation markers
Primary B cells isolated from the bone marrow and spleen of 2 mice were pooled on days 0, 3, 7 and 14 after secondary immunization i.p. (100 uL) with recombinant tetanus toxin heavy chain fragment C (TT, FinaBio; 10 ug). Cell preparation for single-cell sequencing was first performed, which included cell count, washing and resuspension in 1x PBS with 0.04% BSA.  The samples containing single cells in suspension (with an estimated 1600 cells/µl) are combined with barcoded Single Cell VDJ 5' Gel Beads, and Partitioning Oil onto Chromium Next GEM Chip K, generating GEMs. To achieve single cell resolution, cells are delivered at a limiting dilution, such that the majority (~90 – 99%) of generated GEMs contains no cell, while theremainder largely contain a single cell. Immediately following GEMgeneration, the Gel Bead is dissolved and any co-partitioned cell is lysed. Oligonucleotides containing (a) an Illumina R1 sequence (read 1 sequencing primer), (2) a 16 nt 10x Barcode, (3) a 10 nt unique molecularidentifier (UMI), and (4) 13 nt template switch oligo (TSO) are released and mixed with the cell lysate and a Master Mix containing reverse transcription (RT) reagents and poly(dT) RT primers. Incubation of theGEMs produces 10x Barcoded, full-length cDNA from poly-adenylated mRNA. The GEMs are then broken and the 10x Barcoded first-strand cDNA is purified and amplified via PCR with primers against common 5' and 3' ends. This generates sufficient material to construct multiple libraries from the initial single cells. The 10x libraries were prepared for sequencing using the Chromium Single Cell 5' Reagent Kits v2 (Dual Index) (10x Genomics), following the manufacturer's recommendations. Shortly, the amplified cDNA generated previously is used to generate 5ʹ Gene Expression library. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size prior to library construction. P5, P7, i5 and i7 sample indexes, and Illumina R2 sequence (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and sample Index PCR. The final gene expression libraries contain the P5 and P7 priming sites. No VDJ libraries were generated for this porject.