GSM4476751: ATAC_Villus_rep1; Mus musculus; ATAC-seq
Freshly harvested duodenum was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 3 mM EDTA in PBS for 5 mins, 10 mins and 40 mins at 4 °C (refresh EDTA/PBS every time). The tissue was then violently shaken to release the epithelium from underlying muscular tissue. The intestine pieces were discard and the supernatant was collected as whole epithelium fraction. 70-μm cell strainer was used to separate the villi and crypt fractions. Villi were collected from the top of a 70-μm cell strainer, while crypts passed through. Cells were pelleted by centrifugation at 170g at 4 °C, and followed by cold PBS wash. Duodenal villi and crypts were treated with pre-warmed 0.25% Trypsin for 8 mins at 37°C on a vortex station, neutralized with 10% FBS, and passed through a 40-μm cell strainer to obtain single cells for ATAC-seq. 50,000 cells were used for ATAC-seq. Briefly, cells were centrifuged at 500g for 5mins at 4°C, and resuspended in ice-cold lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, 3 mM MgCl2 and 0.1% NP-40), followed by centrifugation at 500 g for 10 minutes at 4°C. The isolated nuclear pellets were incubated with a 50 μl reaction of Nextera Tn5 Transposase (Illumina FC-121-1030) for 30 min at 37°C. The transposed chromatin was purified with QIAquick PCR Purification Kit (QIAGEN) and PCR amplified with high-fidelity 2X PCR Master Mix (New England Biolabs M0541). The optimum number of additional cycles was based on 1/3 of the maximum fluorescent intensity. The PCR amplified libraries were purified, fragment size was selected using Pippin Prep. NEBNext High-Fidelity 2X PCR Master Mix (New England Biolabs)