title: GSM4171675: FACS Sorted cells from the small intestine, WT3; Mus musculus; RNA-Seq
library_construction_protocol: The ileum (distal 14cm of the small intestine) was dissected from mice. Mesenteric fat tissue and Peyer's patches were carefully removed from these tissues. Intestinal tissue was opened and extensively cleaned of fecal matter. Following, this tissue was sequentially treated with HBSS 1X (1 mM DTT) at 37°C for 10 min with gentle shaking (200rpm), and twice with 5 mM EDTA at 37°C for 10 min to remove epithelial cells. The remaining tissue was then minced with a scissor and dissociated in RPMI containing 10% FBS, Dispase (0.05 U/ml; Worthington), collagenase (1 mg/ml collagenase II; Roche) and DNase I (100 μg/ml; Sigma) at constant shaking at 37°C for 45 min (175rpm). The digested tissue was then filtered through a 70μm strainer to remove large debris. Viable Lamina Propria Lymphocytes (LPLs) were collected at the interface of a 40%/80% Percoll/RPMI gradient (GE Healthcare). Cells were stained for surface markers. DAPI (ThermoFisher) was used to exclude dead cells. The following monoclonal antibodies were purchased from eBiosciences, BD Pharmingen or BioLegend: CD3, CD45.1, CD45.2, TCRβ, CD11c, CD14, CD19, TCRβ, TCRγ, NK1.1, CD127, CD90.2, CCR6. Sorting was performed on an Aria II (BD Biosciences). RNA quantity and quality were determined using Agilent's RNA 6000 Pico Chip RNA-seq libraries were prepared with the Low input Clontech SMART-Seq HT with Nxt HT. The sequencing was performed using the Nextseq 500 High output kit v2. RNA-seq libraries were prepared and sequenced by the Genome Technology Core at New York University School of Medicine.
attributes.tag: GEO Accession
attributes.value: GSM4171675