title: GSM3891776: VIRF3_1: vIRF3 Rep. 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were crosslinked with 1% formaldehyde in PBS for 10 min and quenched with 0.125 M glycine for 5 min at room temperature. All buffers were supplemented with 1X protease inhibitor cocktail (Roche) and 1 mM phenylmethylsulfonyl fluoride. Cells were incubated in 1 mL buffer 1 (50 mM HEPES-KOH, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100) for 10 min on ice. Nuclei were then pelleted at 1,350 g,  for  5 min, and resuspended in 1 mL buffer 2 (10 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA). Pelleted nuclei were lysed in 1 mL buffer 3 (1% SDS, 10 mM EDTA, 50 mM Tris-HCl) by multiple pipetting.  and cChromatin was sheared by sonication using a BioruptorTM (Diagenode). 100 µl 10% Triton X-100 was added to each sample, samples were mixed, and cell debris was removed by centifugation at 20,000 g and, 4°C, , for 10 min.   For each ChIP, chromatin from 1x106 cells was diluted 1:10 in ChIP dilution buffer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, 167 mM NaCl) and incubated with 4 µg of the respective antibody for 16 h rotating at 4°C. Chromatin immunocomplexes were bound to 50 µl BSA-blocked Pierce ChIP-grade Protein A/G Magnetic Beads (Thermo Fisher Scientific, Cat.# 26162) for 1 hr at 4°C.   Beads were washed using increasing salt concentrations: low-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 150 mM NaCl); high-salt buffer (0.1 % SDS, 1 % Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, 500 mM NaCl); LiCl-wash buffer (0.25 M LiCl, 1 % Nonidet P-40, 1 % sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl); with the final two washes with TE buffer without protease inhibitors.   Chromatin was eluted in 210 µl elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) for 30 min at 65°C. De-crosslinking was performed by adding 8 µl 5M NaCl at 65°C overnight. Chromatin was diluted in with 200 µl TE buffer and treated with 8 µl RNAse A (10 mg/ml) for 2 h at 37°C. After the addition of 7µl CaCl2 solution (300 mM CaCl2 in 10 mM Tris-HCl), remaining protein was degraded using 4 µl Proteinase K (40 mg/ml) for 1h at 55°C. DNA was purified by standard phenol/chloroform extraction followed by ethanol precipitation. Samples were resuspended in 55µl 10 mM Tris-HCl. Input DNA from 2.5X105 cells was de-crosslinked and purified similarly to ChIP samples. Sequencing libraries were generated from 2-10 ng ChIP DNA or input using the NEXTflex Illumina ChIP-Seq Library Prep Kit (Bioo Scientific, Cat#5143-02) as per the manufacturer´s instructions.