title: GSM6735800: TotalGC_Bcells_RNA; Mus musculus; RNA-Seq
library_construction_protocol: For flow cytometry and cell sorting, disposable micropestles (Axygen) were used to mechanically disassociate LNs into cell suspensions. Cells from each tissue were resuspended in PBE 1X (PBS supplemented with 0.5% BSA + 1 mM EDTA) and incubated for 30 min on ice with fluorescently-labeled antibodies described in the Key Resources Table. Cells were filtered and washed with PBE again before analysis on BD FACS LSR II or FACS Symphony cytometers and sorting on FACS ARIA II cytometers. Analyses were performed using FlowJo v. 10 software. Total GC B cells were sorted, counted for viability, and immediately subjected to library preparation. We produced gene expression and BCR single-cell libraries using the 10X Chromium Single Cell 5' and V(D)J Enrichment, according to the manufacturer's instructions at the Genomics core of Rockefeller University. The scRNA and BCRseq libraries were sequenced on the Illumina NextSeq500 platform. The sequencing yielded 558 million reads with an average of 194,452 reads per cell using read lengths of R1: 26 bp [16bp cell barcode:10bp Unique Molecular Identifier (UMI)], R2: 57 bp read 2. In addition, single-cell BCR libraries yielded an average sequencing depth of 32,626 reads per cell using read lengths of 151 bp read 1 and 151 bp read 2.