GSM8139435: hFOB 1.19, differentiated 5 days at 39.5⁰C, rep2; Homo sapiens; OTHER
For this protocol, 107 fixed hFOB cells were resuspended in dH2O supplemented with protease inhibitor cocktail and incubated on ice for 10 minutes twice. After setting aside 50μl of cell suspension for pre-digestion QC, the remaining sample was divided into 6 tubes. All incubation reactions were carried out in a Thermomixer (BenchMark) shaking at 1,000 rpm. Samples were pre-digested for 1 hour at 37°C after adding 0.3% SDS, 1x NEB DpnII restriction enzyme buffer and dH2O. We then added a 1.7% solution of Triton X-100 to each tube and continued the incubation an additional hour. In the sample tubes only, we added 10µL of DpnII (NEB, 50 U/μl) and continued the incubation until the end of the day when another 10μl DpnII was added to each sample to digest overnight. The next morning, we added another 10μl DpnII and incubated for a final 2−3 hours. We removed 100µL of each digestion reaction, pooled them into two 1.5ml tube, and set them aside for digestion efficiency QC. We heat inactivated the remaining samples at 65°C for 20 minutes, before cooling on ice for 20 minutes. We ligated digested samples overnight at 16°C with T4 DNA ligase (HC ThermoFisher, 30U/μl) and 1X ligase buffer. The next day, we spiked an additional T4 DNA ligase into each sample and incubated another few hours. We then de-crosslinked the samples overnight at 65°C with Proteinase K (20mg/ml, Denville Scientific) along with pre-digestion and digestion controls. The following morning, we incubated the controls and ligated samples for 30 minutes. at 37°C with RNase A (Millipore) prior to phenol/chloroform extraction, ethanol precipitation at −20°C, and centrifugation at 4°C and 3000 rpm for 45 minutes to pellet the samples, while controls were pelleted at 14,000 rpm. Pellets were washed in 70% ethanol and centrifuged again as described above. We resuspended the 3C library and control pellets in dH2O and stored both at −20°C. We measured sample concentrations by Qubit and assessed digestion and ligation efficiencies by gel electrophoresis on a 0.9% agarose gel and by quantitative PCR (SYBR green, Thermo Fisher). DNA from each 3C library (10ug) was sheared to an average fragment size of 350bp using a QSonica Q800R (60% amplitude, 30 seconds on / 30 seconds off, 2-minute intervals for 5 total intervals) at 4°C. After shearing, DNA was purified using AMPureXP beads (Agencourt), DNA size was confirmed on a Bioanalyzer 2100 (Agilent) and DNA concentration measured via Qubit. Libraries were prepared for selection using the SureSelect XT Library Kit (Agilent) following the manufacturer protocol and once again bead purified, then checked for size and concentration as described above. One microgram of adaptor-ligated library was hybridized using the SureSelect XT capture kit (Agilent) and our custom-designed 41K promoter Capture-C probe set. After amplification and purification, we assessed the quantity and quality of the captured libraries one final time. We paired-end sequenced all promoter-focused capture-C libraries on the Illumina NovaSeq 6000 platform with 51bp read length.