GSM3132029: T_Gall_1; Mus musculus; RNA-Seq
For single cell epithelial preparations from small intestines, gall bladder, and colon, tissues were flushed with PBS, opened, and rinsed with PBS to remove intestinal contents. Intestinal tissue was cut into 2-5 cm pieces and incubated rocking at 37° C in 15 mL HBSS (Ca+2/Mg+2-free) supplemented with 5mM dithiothreitol (DTT, Sigma-Aldrich), 5% fetal calf serum (FCS, VWR), and 10mM HEPES (Gibco). Tissues were vortexed vigorously and supernatant was discarded. Tissues were then incubated rocking at 37° C in 15 mL HBSS (Ca+2/Mg+2-free) supplemented with 5mM EDTA (Invitrogen), 5% FCS, and 10mM HEPES. Tissues were vortexed thoroughly and released epithelial cells were passed through a 70 μm filter. Tissues were then incubated in fresh EDTA/HBSS solution for 15 minutes, vortexed, and filtered. Supernatants were pooled and washed once before staining for flow cytometry. Cells were sorted directly into lysis buffer from the Dynabead mRNA Direct Purification Kit (ThermoFisher). Sorted cells were pooled for bulk sequencing and poly A RNA was isolated according to the manufacturer's protocol. amplification protocol: Amplified cDNA was prepared using the NuGen Ovation RNA-Seq system V2 kit, according to the manufacturer's protocol Sequencing libraries were generated using the Nextera XT library preparation kit with multiplexing primers, according to manufacturer's protocol (Illumina)