title: GSM5111723: Mtb K -> LCMV + αIFNAR1 BCR; Mus musculus; RNA-Seq
library_construction_protocol: To create a single-cell suspension, each lung was chopped into small pieces, and incubated in RPMI supplemented with 1.3 mM EDTA at 37℃ on a shaking incubator for 30 min. After the incubation, the small fragments were incubated again in complete medium solution containing 0.1% collagenase type II (Worthington Biochemical, Lakewood, NJ, USA) for 1h. The single-cell suspensions were then filtered through a 40-μm cell nylon mesh strainer, treated with RBS lysis buffer (Gibco) for 3 min, and washed twice with RPMI containing 2% FBS. Lymphocytes from trachea lymph nodes were mashed through a 70- μm cell strainer (BD Falcon), and red blood cell (RBC) lysis was performed using ACK lysing buffer (Gibco Laboratories). For CD45.2+ cell enrichments, suspensions of pulmonary cells were enriched by two-step magnetic cell sorting. Briefly, lung cells were incubated with CD45.2-FITC antibody (Miltenyi Biotec) and washed out. Then, cells were incubated with Anti-FITC microbeads (Miltenyi Biotec) and separated by sequential passage through LS MACS column (Miltenyi Biotec). The positive fraction was mostly composed of CD45.2+ cells. Single-cell sequencing libraries were prepared by using Chromiun 10X Genomics platform with a ChromiumTM Next GEM Single Cell 5' Library & Gel Bead Kit (10X genomics) and ChromiumTM Single Cell V(D)J Enrichment Kit, mouse T cell (10X genomics) according to the manufacturer's instructions.