GSM5324940: V_U_58349_udLN; Mus musculus; RNA-Seq
For bulk RNA sequencing, stromal cells were prepared for cell sorting by mincing thymus, lymph nodes, and spleen with razor blades and then digesting for 30 minutes at 37C in 100ug/mL DNase I (Roche) and Liberase TM (Roche) before filtering through a 70-micron cell-strainer. Density-gradient centrifugation was used to enrich for stromal cells, using a three-layer Percoll PLUS gradient (Sigma) with specific gravities of 1.115, 1.065, and 1.0. Cells isolated from between the 1.065 and 1.0 layer were resuspended in anti-Fc receptor blocking antibody clone 2.4G2 for 10 minutes, then incubated on ice for 30 minutes with antibody cocktail. 4',6-diamidino-2-phenylindole (DAPI) was added to exclude dead cells before sorting on a FACSAriaII (BD Biosciences, San Jose, CA ). CD3- CD19- NK1.1- MHCII+ GFP+ cells were sorted directly into cold RPMI with 2% FBS, washed with PBS twice, and then resuspended in lysis bffer and snap frozen for RNA isolation and sequencing. SmartSeq/NexteraXT bulk RNA-sequencing