title: Illumina HiSeq 4000 paired end sequencing; Single cell RNA-seq and VDJ-seq of B cells from sheep red blood cells-primed WT and Stch cKO mice
design: Single cell RNA-seq and VDJ-seq of B cells from sheep red blood cells-primed WT and Stch cKO mice
library_construction_protocol: Splenic lymphocytes were isolated from 9-week-old female stchf/f wildtype (WT) mice and B-cell specific Stch knocked-out (CD19creItchf/f) mice (3 mice/group) using Ficoll (Ficoll-Paque Plus, GE Healthcre). B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany). Samples were washed three times with PBS. Cells were processed using 10x Genomics Chromium protocols. Mice were euthanized using CO2. Spleens were cut using scissors.  B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany). B cells were not cultured in vitro. Cells were processed using 10x Genomics Chromium protocols. CD19cre mice and Stchf/f mice were developed by Cyagen Co., Ltd (Guangzhou, China). CD19cre mice and SItchf/f mice were mated to get Stchf/f (WT) and CD19creStchf/f (Stch cKO). Splenic lymphocytes were isolated from 7-9-week-old female WT mice and Stch cKO mice (3 mice/group) using Ficoll (Ficoll-Paque Plus, GE Healthcre). B cells were sorted by B220 microbeads (Cat No. 130-049-501, Miltenyi Biotec, Germany). Cells were processed using 10x Genomics Chromium protocols. Data were aligned using STAR with options --outFilterScoreMinOverLread 0.4 --outFilterMatchNminOverLread 0.4, against the C3H/HeJ genome as a reference (ftp://ftp.ensembl.org/pub/release-89/fasta/mus_musculus_c3hhej/dna/Mus_musculus_c3hhej.C3H_HeJ_v1.dna_sm.toplevel.fa.gz).  Sorted cells were loaded onto a 10x Chromium Chip using reagents from the Chromium Single Cell 5' Library and Gel Bead Kit (10x Genomics) according to the manufacturer's protocol. Amplified cDNA 11 was used for both 5' RNA-seq library generation and TCR V(D)J targeted enrichment using the Chromium Single Cell V(D)J Enrichment Kit for Mouse T Cells (10x Genomics). 5' RNA-seq and TCR V(D)J libraries were prepared following the manufacturer's user guide (10x Genomics).