title: GSM6460309: PdgfraLo_CD81+_Duodenum_Rep2; Mus musculus; RNA-Seq
library_construction_protocol: Adult murine tissue was harvested by dividing the small intestine into 5 segments of equal length using the first one as Duodenum, the third as Jejunum and the fifth one as Ileum, intermediate regions discarded. For colonic cells, the proximal half of the colon (approximal 3cm) was used. TIssue was washed and the outer muscle layer was removed with tweezers under a stereomicroscope. Tissue pieces were incubated twice in 10mM EDTA in HBSS  +10mM HEPES + 5%FBS for 30min at 37°C, washed and digested with 3mg/ml collagenase2 for 20min at 37°C. The filtered cell suspension was stained with biotinylated anti-CD81 (eBiosciences 13-0811-81) or control biotinylated IgG for 25min at 4°C, washed and incubated with streptavidin-APC (eBiosciences, 17431782) for 25min at 4°C. Cells were sorted three-way for Pdgfra_eGFP high; Pdgfra_eGFP low CD81+ and Pdgfra_eGFP low CD81-  at the Dana-Farber Flow cytometry core facility using a BD FACS M Aria II instrument. Freshly sorted mesenchymal cells from different regions were used immediately for RNA extraction with RNAeasy Plus micro kit (Qiagen). Samples with RNA-integrity number of >8.5 were processed with SMART-Seq v4 Ultra Low Input RNA library construction kit per manufacturer's recommendation; and sequenced on Illumina Novaseq platform by the molecular biology core facility at Dana-Farber.