title: GSM5639557: mLN1st_2; Mus musculus; RNA-Seq
library_construction_protocol: Isolation of cells from mFAT and SI LP: For isolation of cells from mFAT mice were euthanised and transcardially perfused with 10ml of ice-cold PBS. mFAT was separated from mLN, intestine and omentum, minced and digested in 3ml of low glucose DMEM containing 25mM HEPES, 1% low fatty acid BSA, 2mM L-glutamine (L-glut), 100U/ml Penicillin/Streptomycin (P/S), with the addition of Liberase TL (0.2mg/ml, Roche) and DNase I (0.25mg/ml, Roche) for 30-40min in 37°C rotating. After digestion 2ml DMEM containing 2mM EDTA was added and the suspension filtered through 70um strainer. Cells in SVF were then separated from the adipocyte layer by centrifugation. For isolation of cells from SI LP small intestines were open longitudinally, cleaned, cut into 2cm pieces and placed in RPMI medium containing 3% FBS, 25mM HEPES, 2mM L-glut, 100U/ml P/S, 1mM DTT and agitated for 25min, after which samples were washed 3-4 times with RPMI containing 2mM EDTA. Intestines were digested in RPMI medium containing 25mM HEPES, 2mM L-glut, 100U/ml P/S, Liberase TL As described in CEL-Seq2 protocol (Hashimshony et al. 2016) Adapted from TruSeq Small RNA Library Preparation Protocol