GSM7767344: MEF_caMEK1_Dox_d7_2; Mus musculus; RNA-Seq
Hepatocyte;The liver was perfused with EGTA solution (calcium-free Hank's balanced salt solution (HBSS) (Wako) containing 0.5 mM EGTA (Sigma) and 10 mM HEPES (Nacalai Tesque)), followed by a second perfusion with HBSS containing collagenase D (SIGMA) for bulk RNA-seq or Liberase TM Research Grade (Roche) for scRNA-seq and 10 mM HEPES. After passage through a 70 µm cell strainer, the cells were centrifuged at 50 g for 2 minutes three times to enrich for hepatocytes.  Colonic epithelial cells; The colon was dissected, cut open longitudinally and rinsed with ice-cold HBSS three times. The total colon without the caecum was used. The tissue was incubated for 30 min at 37 °C in isolation buffer (HBSS containing 10 mM HEPES (Nacalai Tesque), 5 mM EDTA (GIBCO) and 1 mM DTT (Sigma)) and then shaken vigorously for 30 sec to separate the crypt from the stromal tissues. After passage through a 70 µm cell strainer, the crypt suspension was centrifuged at 800 rpm for 3 min. The supernatant was decanted, and the crypt pellet was subsequently resuspended in 0.25% trypsin/1 mM EDTA and incubated for 15 min at 37 °C. The cell suspension was centrifuged at 3000 rpm for 5 min. Dissociated hepatocytes and crypt cells were passed through a 40 µm cell strainer and sorted with a FACSAria II (BD Bioscience) machine using a 130 µm nozzle. Dead cells were gated out by DAPI incorporation (100 ng/mL for crypt cells and 500 ng/mL for hepatocytes). To enrich for hepatocytes, we used 2 FSC-neutral density (ND) filters and 10-SSC ND filters. Ccells were sorted into RNAprotect Cell Reagent (QIAGEN) and stored at -80 °C until RNA extraction. Total RNA was extracted using the RNeasy plus Mini or Micro Kit (QIAGEN) according to the manufacturer's protocol. RNA was quantified with a NanoDrop instrument (Thermo Scientific). Total RNA (in vitro sample; 100 ng, FACS-sorted samples; 50 ng) was prepared for library construction. High-quality RNA samples (RNA Integrity Number value > 8, as determined by Bioanalyzer) were used for library construction. RNA-seq libraries were generated using the NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEB). The number of PCR cycles was minimized to avoid skewing the representation of the libraries. RNA-seq libraries were sequenced on a NextSeq 500 (Illumina) as 76-bp single-end reads.