GSM9476720: pool616-1_2_GEX; Mus musculus; RNA-Seq
5 minutes prior to euthanasia, mice were injected with 1µg α-CD45-BUV395 or α-CD45-APC in the retro-orbital cavity. Lungs and mediastinal lymph nodes (medLN) were collected from euthanized mice. Lung tissue was digested with 1.5mg/mL collagenase (Milipore Sigma) and 10µg/mL DNAse I (Milipore Sigma) in Mg2+, Ca2+ free Hank's balanced salt solution (HBSS, Gibco) with 10% fetal bovine serum (FBS, Sigma) for 1 hr at 37°C or 30 min in a shaking incubator at 1000 rpm. Lungs and medLN were then ground into a 40µm filter to generate a single cell suspension. Prior to staining, cells were blocked with Fc block for 10 minutes and then stained with fixable LiveDead Aqua (ThermoFisher) to identify dead cells, surface antibodies, hashtag oligos for each mouse and PR8-HA and Cal09-HA B cell tetramers for 20 minutes at 4C. Cal09-HA and PR8-HA tetramers were bound to SA-Oligo tagged-PE and SA-BV711 to gate out SA-PE specific B cells from the lungs and medLN. All cells from each mouse were combined into a single pool per tissue and sorted to enrich for tetramer-specific cells gated as live CD45-unlabeled B220+ HA-Tetramer++. Enriched HA-tetramer specific B cells were run on 10x Chromium with 5' GEM-X kit to isolate single cells, and libraries generated for gene expression, BCR sequencing, and feature barcode quantification.