GSM3941327: RNASeq_E13.5_Stomach_Replicate #2; Mus musculus; RNA-Seq
For E13.5 mouse experiments, ShhCre; R26YFP/+ embryos were used for downstream high-throughput experiments. After dissection, the stomach and intestinal organs were isolated, separated, washed in cold PBS and digested in 4ml of a 3:1 solution of Trypsin LE: 1x PBS at 37˚C. After 10 minutes, tissue was vigorously pipetted, until the organs broke down into a single cell suspension, which was confirmed using brightfield microscopy. The enzymatic reaction was then inhibited with 10% FBS. After washing cells in cold PBS, the single cells were re-suspended in 400µl of 1:5000 Sytox Blue (ThermoFisher Scientific, S34857) in cold 1x PBS, filtered through a 30µm mesh and submitted for sorting. Sorted cells were washed again and processed for ATAC-seq (50,000 cells from 3-4 YFP+ embryos) using the previously published protocol (44).    For E16.5 experiments, whole organ digestion using Trypsin was detrimental to epithelial cell viability; thus, an alternative isolation approach was employed. Given strict cell numbers are required for ATAC-seq, gut epithelium was isolated from ShhCre;R26YFP/+ embryos prior to single cell dissociation. In more detail, the E16.5 embryonic stomach, the proximal small intestine and the colon were dissected and opened longitudinally using a 0.5 cm tungsten needle. The forestomach was separated from the hindstomach along the squamous-glandular border under a dissecting microscope, and incubated in Dispase solution for 15 minutes at 37˚C. Following, the epithelial layer was manually separated from the mesenchyme and flash frozen for RNA isolation or collagenase digested for sorting. The hindstomach, proximal small intestine and colon were incubated in 10mM EDTA, 5mM EDTA and 5mM EDTA in 1x PBS, respectively, for 30-40 minutes at 4˚C on a shaker. Tissues were then moved to fresh 1x PBS and shaken vigorously for 2-3 minutes, or until epithelial glands/villi/crypts were observed in suspension. Isolated epithelial cells were immediately frozen and RNA isolation was conducted using an RNA isolation kit (Qiagen, Cat No. 74104). To prepare for sorting for ATAC-seq experiments, the fresh epithelial pellets of the forestomach, hindstomach, intestine and colon were then digested in collagenase-based solution for 20 mins at 37˚C. The resulting single cell suspension was washed in cold 1x PBS, re-suspended in 500µl of 1:5000 Sytox Blue and submitted for sorting. Sorted cells were then used for ATAC-seq using the same protocol as mentioned above. Whole transcriptome sequencing libraries were prepared using TruSeq® Stranded mRNA Library Prep Kit (RS-122-2101, Illumina, San Diego, CA, USA) by following the manufacturer's instructions.