GSM4816232: ATACseq_whole_tissue_E16_5_mCtrl_rep2; Mus musculus; ATAC-seq
Mice were euthanized with CO2, perfused via the left ventricle of the heart with 10 ml of PBS, siLP, liLP, lung and skin were collected and placed in cold complete medium (RPMI 1640 supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM nonessential amino acids, 20 mM HEPES, 50 mM b-mercaptoethanol, 100 U/mL penicillin, 100 mg/mL streptomycin). For siLP and liLP, Peyer's patches and mesenteric adipose tissue was removed. Tissues were open and wash in cold PBS to remove feces. Tissues were cut into 1-2 cm segments and treated with complete RPMI medium containing 5mM EDTA and 0.145 mg/ml dithiothreitol for 20 min at 37°C with constant stirring. Tissues were shake vigorously for 1 min, further digested with 10 ml 500 mg/ml DNase I (Sigma-Aldrich) and 100 mg/ml Liberase TL (Roche) with continuous stirring at 37°C. Whole tissue RNA-seq: 3 cm of intestinal segments from duodenum, jejunum and ileum was collected and extensively washed with PBS to clean out feces. Samples were homogenized by beads beating with TRIzol in tubes containing 28mm beads and TRIzol (Thermofisher). RNA was extracted according to TRIzol protocol. cDNA was synthesized with the Ultralow V4 kit (Clonetech) and sequencing libraries were subsequently prepared with the Nextera XT DNA prep kit (Illumina). Epithelial cells RNA-seq: Epithelial cells were sorted into RNA lysis buffer and RNA was extracted according to manufactoring instructions (Qiagen). RNA library was prepared using the NuGen mRNA-seq kit with mouse probes. Epithelial cells ATAC-seq: Cells were pelleted by centrifugation at 500 g for 5 min at 4°C. Supernatant was removed, and cell pellet was resuspended with 45ul transpose mixture (25 ml Tagment DNA Buffer, 2.5 ml Tagment DNA enzyme, 0.5 ml 1% digitonin and 17 ml H2O ) and incubated for 30min at 37°C with agitation at 300 rpm. Transposed DNA was purified using QIAGEN MinElute Reaction Cleanup Kit and purified DNA was eluted in 10ul elution buffer. Transposed fragments were amplified with 10-15 cycle by PCR based on amplification curve. Once the libraries were purified by QIAGEN PCR cleanup kit, sample were further sequenced single-end on HiSeq 2500.
GEO Accession
GSM4816232