title: RNA-Seq analysis of LYN deficient stromal cells sorted from murine spleens
design: RNA-Seq analysis of LYN deficient stromal cells sorted from murine spleens
library_construction_protocol: Spleens were isolated from Lynwt/wt and Lyn-/- mice and digested according to a modified established protocol. In brief, 1ml of digestion cocktail (0.4mg/ml Collagenase P, 1.2U/ml Dispase and 50µg DNase I in 1640 RPMI with 1% P/S and 10% FBS) was injected per spleen and incubated for 5 min at RT. Then, spleens were mechanically minced and incubated for 30 minutes in additional 2ml of digestion cocktail at 37°C under repeated mixing. Cells were filtered through an 100µm filter, erythrocytes were lysed in ACK-buffer and subsequently washed in PBS. Resulting single cell suspension was labelled with 7AAD and fluorescent antibodies as indicated in Table S1. Splenic fibroblasts were sorted via FACS as 7AAD- CD45- CD71- CD31- CD29+ CD54+ cells at the FACS & Imaging Core Facility, Max-Planck Institute for Biology of Aging.  mRNA was isolated using the RNeasy plus kit (Qiagen) according to the manufacturer's instructions and RNA quantity and quality was checked by NanoDrop1000 (Thermo Scientific) spectrometric measurement. Samples were processed and sequenced PE100, 35M reads per sample in the Cologne Center for Genomics according to in-house standards.