GSM5321664: Germinal Center B cell Donor 103 (Capture-C); Homo sapiens; OTHER
Tonsillar mononuclear cells were isolated from tissues by mechanical disruption (tonsils were minced and pressed through a 70-micron cell screen) followed by Ficoll-Paque centrifugation For each library, 10e7 fixed cells were thawed at 37ºC, followed by centrifugation at RT for 5 mins at 14,000 rpm. The cell pellet was resuspended in 1 mL of dH2O supplemented with 5 μL 200X protease inhibitor cocktail, incubated on ice for 10 mins, then centrifuged. The cell pellet was resuspended to a total volume of 650 μL in dH2O. Both 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 protocol and our custom-designed 41K promoter Capture-C probe set. Promoter Focused Capture C