title: GSM8769017: Lung memory B cells on day 21 (biol rep 2); Mus musculus; RNA-Seq
library_construction_protocol: Mice were injected with fluorophore-conjugated anti-CD45 3 minutes before sacrifice to label circulating cells. Cells were isolated from the lungs by mincing tissue and digesting in 2 mL solution of 750 U/mL collagenase I and 200 U/mL DNase I in RPMI for 45 minutes at 37 C. Digested lungs were passed through 70 micron cell strainers, diluted with 10 mL of 30% Percoll in RPMI and centrifuged for 930 g for 10 minutes with no brake. Red blood cells were lysed for 5 mins at RT in ACK lysis buffer and cells were washed and resuspended in PBS + 2% FBS. Cells were isolated from mediastinal lymph nodes by mushing through 70 micron cell strainers followed by washing and resuspending in PBS + 2% PBS. Isolated cells were stained with anti-CD19, anti-CD38, anti-GL7, and anti-IgD antibodies and resuspended in PBS + 2% FBS + DAPI (1:2000).Cells were sorted as live (DAPI negative), non-circulating (i.v. anti-CD45 negative), B220+ CD38+ GL7- IgD-. Sorted cells were washed and resuspended in PBS without calcium or magnesium + 0.05% BSA. Libraries were prepared using 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kits v3 (Dual Index). 2 biological replicates per time point (day 21 and day 77) were included and 2,500 cells per sample were loaded into individual wells of Chromium Next GEM Chip G and partitioned into Gel Bead-In Emulsions (GEM) and barcoding by 10X Genomics Chromium Controller. Reverse transcription, cDNA amplification, and library construction were performed according to manufacturer's instructions including addition of Illumina i5 and i7 index sequences.