GSM5018029: MSC dervived Adipocyte donor 8013; Homo sapiens; ATAC-seq
Cells were fixed after 2 weeks of adipogenic induction. Cells were collected and single-cell suspension were made with aliquots of 10^7 cells in 10 ml media (e.g. RPMI + 10%FCS). 540 µl 37% formaldehyde was added and incubated for 10 min at RT. The reaction was quenched by adding 1.5 ml 1 M cold glycine (4 °C) for a total volume of 12 ml. Fixed cells were centrifuged at 1000 rpm for 5 min at 4 °C and supernatant removed. The cell pellets were washed in 10 ml cold PBS (4 °C) followed by centrifugation. Supernatant was removed and cell pellets were resuspended in 5 ml of cold lysis buffer (10 mM Tris pH8, 10 mM NaCl, 0.2% NP-40 (Igepal) supplemented with protease inhibitor cocktail). Resuspended cells were incubated for 20 min on ice, centrifuged as above, and the lysis buffer removed. Finally, cell pellets were resuspended in 1 ml fresh lysis buffer, transferred to 1.5 ml microfuge tubes and snap frozen (ethanol/dry ice or liquid nitrogen). Cells were stored at −80 °C until they were thawed for 3C library generation. Samples underwent a pre-digestion incubation with the addition of 0.3% SDS, 1x NEBuffer DpnII, and dH2O for 1hr at 37ºC in a Thermomixer shaking at 1,000rpm. A 1.7% solution of Triton X-100 was added to each tube and shaking was continued for another hour. After the pre-digestion incubation, 10 μL of DpnII (was added only to each sample tube, and continued shaking until the end of the day. An additional 10 µL of DpnII was added to each digestion reaction and digestion continued overnight. The next day, another additional 10 µL of DpnII was added and the incubation continued for another 2-3 hours. 100 μL of each digestion reaction was then removed, pooled into one 1.5 mL tube. The remaining samples were heat inactivated at 65°C for 20 min at 1000 rpm in a Thermomixer, and cooled on ice for 20 additional minutes. Digested samples were ligated with 8 μL of T4 DNA ligase (HC 30U/uL) and 1X ligase buffer at 1,000 rpm overnight at 16°C in a Thermomixer. The next day, an additional 2 µL of T4 DNA ligase was spiked into each sample and incubated for another few hours. The ligated samples were then de-crosslinked overnight at 65°C with Proteinase K along with the pre-digestion and digestion controls. The following morning, both controls and ligated samples were incubated for 30 min at 37°C with RNase A, followed by phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation at -20°C. The 3C libraries were centrifuged at 3000 rpm for 45 min at 4°C 14,000 rpm, to pellet the samples. DNA pellets were resuspended in 70% ethanol and again centrifuged as described above. The 3C library pellets were resuspended in 300 μL dH2O and stored at −20°C. 10 μg of isolated DNA from 3C libraries of each library was sheared in dH2O using a QSonica Q800R to an average fragment size of 350bp.  After shearing, DNA was purified using AMPure XP beads. One microgram of adaptor-ligated library was used as input for the SureSelect XT capture kit using manufacturer's protocol and our custom-designed 41K promoter Capture-C probe set.