GSM8464398: Adult Infected Rep 1; Mus musculus; RNA-Seq
Brachial, axial, and popliteal LNs were harvested from infected mice at 2 days post infection (DPI). Inguinal, brachial, and axial LNs were harvested from naïve mice at this same time point as well. Lymph nodes were digested using an enzymatic solution of collagenase IV, dispase, and DNAse I, from which single cell suspensions were generated . To isolate LNSCs from the lymph node cellular suspensions, cells were subjected to a CD45 magnetic microbead negative selection. The CD45-negative cells were then incubated with biotinylated anti-CD31 and anti-gp38 antibodies, and subjected to a CD31/gp38 magentic microbead positive selection to further enrich for LNSCs that expressed CD31 and/or gp38. The resulting LNSCs were then resupended in DMEM with 10% FBS for further scRNA-seq processing. Single-cell suspensions of adult and old LNSCs from naïve and infected lymph nodes were filtered, counted, diluted to optimal range using DMEM supplemented with 10% FBS, and loaded into the Chromium Controller  to capture between 5,000 and 10,000 cells per group, depending on sample yield. Samples were then processed using the Chromium Single Cell 3' Kit v3 or v3.1, per the manufacturer's user guide. Library quality control was performed using the Qubit fluorometer and the Agilent TapeStation . Libraries were pooled and sequenced on the Illumina NovaSeq 6000 PE.