title: GSM8465941: [BCR_PC_S9]; Mus musculus; RNA-Seq
library_name: GSM8465941
library_construction_protocol: cells from draining lymph nodes were washed and resuspended in FACS buffer (PBS containing 5% FCS and 2 mM EDTA) at a concentration of 108 cells/ml. Samples from different mice and different time points were processed separately using cell hashing as described54. Cells were individually stained with distinct barcoded anti-mouse CD45 antibodies (in-house conjugated) in FACS buffer for 30 minutes on ice. Subsequently, cells were washed and stained with a mix of primary antibodies, then Live/Dead Fixable Aqua Dead Cell Stain (Thermofisher). Live cells of interest (IgDneg eYFP+ PC and B cells) from each sample were bulk-sorted using a BD Influx cell sorter. PC and non-PC were captured in distinct wells for droplet-based scRNA-seq to maximize the recovery of BCR sequence information from non-PC. Within each fraction, cells from different samples were pooled and loaded for the 10x Genomics Single Cell 5' v2 workflow. 10x Genomics 5'-end library preparation for gene expression, BCR and hashtag oligonucleotides Libraries were prepared according to the manufacturer's instructions with modifications for generating B cell receptor (BCR) sequencing libraries. Following cDNA amplification, SPRI select beads were used to separate the large cDNA fraction derived from cellular mRNAs (retained on beads) from the hashtag oligonucleotide (HTO)-containing fraction (in supernatant). For the mRNA-derived cDNA fraction, 50 ng were used to generate the transcriptome library, and 10-20 ng were used for BCR library construction.
identifiers.namespace: GEO
identifiers.id: GSM8465941_r1