title: GSM8170511: SGBS Differentiated replicate 1; Homo sapiens; OTHER
library_construction_protocol: For each library, 10^7 fixed cells were thawed at 37C, followed by centrifugation at RT for 5 mins at 1845 xg. The cell pellet was resuspended in 1 mL of dH2O supplemented with 5 uL 200X protease inhibitor cocktail, incubated on ice for 10 mins, then centrifuged. The cell pellet was resuspended to a total volume of 650 uL in dH2O. Samples underwent a pre-digestion incubation in a Thermomixer (BenchMark) with the addition of 0.3%SDS, 1x NEB DpnII restriction buffer, and dH2O for 1hr at 37ºC shaking at 1,000 rpm. A 1.7% solution of Triton X-100 was added to each tube and shaking was continued for another hour. After pre-digestion incubation, 10 ul of DpnII (NEB, 50 U/µL) was added to each sample tube only and continued shaking until the end of the day. An additional 10 µL of DpnII was added to each digestion reaction and digested overnight.  The next day, a further 10 µL DpnII was added and continue shaking for another 2-3 hours.  The samples were incubated at 1000 rpm in a MultiTherm for 20 min at 65°C to inactivate the DpnII, and cooled on ice for 20 additional minutes. Digested samples were ligated with 8 uL of T4 DNA ligase (HC ThermoFisher, 30 U/µL) and 1X ligase buffer at 1,000 rpm overnight at 16°C in a MultiTherm. The next day, an additional 2 µL of T4 DNA ligase was spiked in to each sample and incubated for another few hours. The ligated samples were then de-crosslinked overnight at 65°C with Proteinase K (20 mg/mL, Denville Scientific) along with pre-digestion and digestion control. The following morning, both controls and ligated samples were incubated for 30 min at 37°C with RNase A (Millipore), followed by phenol/chloroform extraction, ethanol precipitation at -20°C, the 3C libraries were centrifuged at 1000 xg for 45 min at 4°C to pellet the samples. The controls were centrifuged at 1845 xg. The pellets were resuspended in 70% ethanol and centrifuged as described above. The pellets of 3C libraries and controls were resuspended in 300uL and 20μL dH2O, respectively, and stored at −20°C. Sample concentrations were measured by Qubit. Digestion and ligation efficiencies were assessed by gel electrophoresis on a 0.9% agarose gel and also by quantitative PCR (SYBR green, Thermo Fisher). The HepG2 libraries were prepared following the Arima Promoter Capture HiC protocol, using two restriction enzymes (targeting ^GATC and G^ANTC as restriction sites). For Promoter focused Capture C, isolated DNA from 3C libraries was quantified using a Qubit fluorometer (Life technologies), and 10 μg of each library was sheared in dH2O using a QSonica Q800R to an average fragment size of 350bp 26,28–31.  QSonica settings used were 60% amplitude, 30s on, 30s off, 2 min intervals, for a total of 5 intervals at 4 °C. After shearing, DNA was purified using AMPureXP beads (Agencourt). DNA size was assessed on a Bioanalyzer 2100 using a DNA 1000 Chip (Agilent) and DNA concentration was checked via Qubit. SureSelect XT library prep kits (Agilent) were used to repair DNA ends and for adaptor ligation following the manufacturer protocol. Excess adaptors were removed using AMPureXP beads. Size and concentration were checked by Bioanalyzer using a DNA 1000 Chip and by Qubit fluorometer before hybridization. One microgram of adaptor-ligated library was used as input for the SureSelect XT capture kit using manufacturer protocol and our custom-designed 41K promoter Capture-C library. The quantity and quality of the captured library was assessed by Bioanalyzer using a high sensitivity DNA Chip and by Qubit fluorometer. SureSelect XT libraries were then paired-end sequenced either the Illumina HiSeq 4000 or on Illumina NovaSeq 6000 platform (51 bp read length). EndoC-BH1, SGBS_undiff, and SGBS_diff libraries were generated in triplicate, while HepG2 were generated in duplicate.