GSM8971809: Spleen, WT, Isotype, BCR-seq; Mus musculus; OTHER
Lymph nodes and spleens were harvested from mice and placed in RPMI-1640 media with 2% fetal bovine serum (FBS). The organs were gently crushed against a 100-micron filter before digestion in collagenase IV (1 mg/ml) and DNAse I (0.2 mg/ml) for 30 minutes at 37C in 5% CO2 to obtain a single cell suspension. Cells were strained through a 100-micron filter, washed with 5 ml of cold FACS buffer consisting of 1% FBS, 2 mM EDTA, and 0.2% sodium azide in HBSS. For the spleen, red blood cell lysis was performed for five minutes. Live photoconverted red+Cd45+ cells were selected by flow sorting. Sorted cells were pooled from 3 mice for each of the 4 experimental cohorts The 10x Genomics Chromium Single Cell 5' Library and Gel Bead Kit was used. We sequenced 5' RNA expression, TCR sequencing, and BCR sequencing as per the manufacturer's protocol.