GSM8011745: Embryo, day13.5, replicate 3, scRNAseq; Mus musculus; RNA-Seq
For samples from E12.5 to E19.5, isolated intestinal samples were washed in cold Dulbecco's phosphate-buffered saline (DPBS, Gibco, C14190500CP) to remove intestinal contents. The E19.5d tissues were opened longitudinally and sliced into small fragments for the following digestion. The embryonic (E12.5d, E13.5d, E14.5d, and E15.5d) tissues were directly pooled into the tube with collagenase I (Gibco, 17100-017) and elastase (Sigma, E2058) to perform digestion at room temperature to obtain cell mixture. The cell mixture was then passed through a 40-µm filter to obtain the single-cell suspension. The obtained single-cell suspension was centrifuged at 300g for 10 min, and incubated with ACK lysis buffer (Gibco, A10492-01) for 1 min on the ice. Another centrifugation at 300g for 10 min was performed and the supernatant was removed. The final fraction was mixed in suitable DPBS with 0.05% BSA and Propidium Iodide. For Lmo3_E19.5 samples, small intestines were collected at E19.5, minced into small pieces, and processed in a solution composed of DNaseI (Merk, 10104159001), and Y27632 (R&D Sytems, 1254/50 ,5000X) in TrypLE (Gibco, 12605-010) for about 15min in 37℃ on 150rpm shaker. The remaining tissues were further digested in digestion buffer containing 1.2mg/ml Collagenase (Sigma, C2139-1G), 1mg/ml Dispase II (Gibco, 17105041), 0.1mg/ml DNase I, and Y27632 in Hank's balanced salt solution (HBSS. Gibco 14170-112) for 20min in 37℃, 150rpm shaker. Tissues were resuspended every 6min through pipetting. The reaction was stopped by adding 3-volume of wash buffer containing 5% FBS (Gibco, 16000-044), Gentamicin / Amphotericin (Gibco, 50-0640, 500X), Penicillin / Streptomycin B (Gibco, 15140-122,100X), HEPES (Sigma, H0887-100ML,100X), Y27632 in F12 culture medium (Gibco, 11765-054). The lysates were passed through 70-μm filters, centrifuged at 300g for 8 min, and resuspended in the wash buffer. 1ml ACK lysis buffer was added to remove the red blood cells on ice for 4min. The remaining cells were washed twice with wash buffer and filtered again with 40-μm Millipore. The final fraction was resuspended into the wash buffer with 1ug/ml DAPI. Cell suspensions with at least 80% cell viability were diluted to the appropriate concentration and loaded on a 10x Genomics Chromium Single Cell Controller (Pleasanton, CA) with a target of about 5000 cells per sample. Single-cell library preparation was completed using the 10x Genomics Chromium Single Cell 3' Library & Gel Bead Kit v2 strictly following the manufacturer's protocol.