GSM3956382
GSM3956382: Juvenile Colon ATAC-seq replicate 1; 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 discarded 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. Duodenum 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. Whole duodenal epithelium fraction was used for juvenile ATAC-seq samples, whereas isolated duodenal villi and crypts were used for adult and geriatric samples. Freshly harvested geriatric proximal colon (combined from 2 mice) was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in 5 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 muscular intestine pieces were discarded and the supernatant was collected as colon epithelium fraction. Cells were pelleted by centrifugation at 170g at 4 °C, and followed by cold PBS wash. Colon epithelial cells were treated with pre-warmed 0.25% Trypsin for 8 mins at 37°C on a vortex station (speed set between 6 and 7), neutralized with 10% FBS, and passed through a 40-μm cell strainer to obtain single cells for ATAC-seq. Freshly harvested juvenile (combined from 6 pups)/adult colon (combined from 2 mice) was flushed with cold PBS, opened longitudinally, cut into 1 cm pieces, and then rotated in EDTA chelation buffer (43.4 mM sucrose, 54.9 mM D-sorbitol, 0.5 mM DL-dithiothreitol and 2 mM EDTA in PBS) for 1 hour at 4 °C. After PBS wash, the tissue pieces were then rotated in digestion buffer (2.5% FBS, 200 U/ml type IV collagenase and125 μg/ml type II dispase in DMEM) for 2 hours at 37 °C. The tissue was then violently shaken to release the epithelium from underlying muscular tissue. The intestine pieces were discarded and the supernatant was collected as colon epithelium fraction. Cells were pelleted by centrifugation at 300g at 4 °C, and followed by cold PBS wash. Cells were resuspended in MACS buffer (0.5% FBS, 2mM EDTA in PBS), and passed through a 70-μm cell strainer. Cells were stained with the anti-CD326 (EpCAM) magnetic microbeads antibody (Miltenyi Biotec, 130-105-958) for 40 min on ice. To obtain single magnetic antibody conjugated EpCAM positive epithelial cells, stained cells were passed through a 40-μm cell strainer and then collected over a MS column (Miltenyi Biotec, 130-042-201) in a magnetic field. After washing with MACS buffer, EpCAM positive epithelial cells were used for ATAC-seq. In all cases, 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. One sample from the geriatric colon couldn't be processed due to low amount of DNA recovered. For other samples, the PCR amplified libraries were purified, fragment size was selected using Pippin Prep, and sequenced on Illumina NextSeq 500 cycle mid output.
GEO
GSM3956382