GSM4907347: IQ3pos_1910A; Mus musculus; RNA-Seq
Isolated mouse stomach was chopped into 2 mm2 pieces and incubated with chelating buffer (5 mM EDTA and 0.52 mM DTT in PBS) for 2 h at 4oC on a shaking platform, then gastric units were released by vigorous shaking in cold dissociation buffer (54.8 mM D-sorbitol and 44 mM Sucrose in PBS). Isolated mouse gastric units were incubated in TrypLE Express (Thermo Fisher Scientific) with Y-27632 (Merck) for 12 minutes at 37oC with repetitive pipetting for digestion into single cells. The cells were passed through a 30 μm cell strainer (Miltenyi Biotech) and labeled with antibodies in HBSS with 2% FBS. Antibodies for the flow cytometry are as follows: anti-EpCAM APC/Cy7-conjugated (BioLegend). Dead cells were excluded by DAPI staining, and labeled cells were sorted by using FACSAria II (BD Bioscience). RNA in FACS-sorted Iqgap3high (IQ3pos) and Iqgap3low/neg (IQ3neg) cells of Iqgap3-2A-tdTomato mice was extracted using NucleoSpin RNA XS (MACHEREY-NAGEL) by following the manufacturer's manual. RNA were sent to Beijing Genomics Institute (BGI) for PCR amplification, transcriptome library preparation and sequencing. SMART-SeqII