title: GSM5107543: NF2-DMSO rep 1; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% formaldehyde for 10', R.T. Cells were incubated with 125 mmol/L glycine for 5' then washed (2X) with cold PBS, centrifuged and lysed in 2 ml ChIP buffer (150 mmol/L NaCl, 1% Triton X-100, 5 mmol/L EDTA, 10 mmol/L TrisCL pH7.5, 0.5 mmol/L DTT, 0.5% SDS) by rocking for 10' at 4°C.  DNA was fragmented by sonication to 0.3-0.5 kb DNA fragments (Misonix S-3000, 4oC water bath, 30 minutes (30 seconds on, 30 seconds off, output 8).  Input DNA (7X10^4 cell equivalent) was removed from fragmented DNA and stored at -80°C.  Anti-BRD4 antibody (3 ug/ per condition) (BL-149-2H5, Bethyl Laboratories) and Dynabeads Protein G (90 ul/ per condition) were added to lysate and incubated overnight at 4°C with rocking.  Beads were washed with buffer 1 (150 mmol/L NaCl, 20 mMol/L TrisCl pH 8.0, 5 mmole/L EDTA, 65% w/v sucrose, 1% Triton-X-100, and 0.2% SDS) and then washed with TE buffer.  ChIP and input DNA were resuspended in TE/1% SDS and incubating at 65°C for 18 hours.  SDS was diluted with TE and treated with RNase A (0.2 mg/ml) at 37°C for 2 hours, treated with proteinase K (0.2 mg/ml) 65°C for 2 hours, then DNA was extracted with phenol chloroform isoamyl alcohol and resuspended in TE. The ChIP-seq libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Ilumina (E7645S, New England Biolabs, Inc. Ipswich, MA, USA).  Library preparation followed these steps; end repair, 5' phosphorylation, dA-tailing, adapter ligation, U excision, cleanup of adaptor-ligated DNA without size selection, and library amplification.  Twelve to 13 cycles were followed by clean-up with 1X Agencourt AMPure XP.  The quality of the libraries was assessed using  Agilent High Sensitivity DNA Kit (5067-4626) on the Agilent 2100 Bioanalyzer and were quantified using NEBNext Library Quant Kit for Illumina (E7630S, New England Biolabs).  Libraries were sequenced using the NextSeq 500 High Output v2 Kit (75 cycles, FC-404-2005) on the NextSeq 500 Platform (Illumina).