GSM4928575: LuCaP_93_H3K27ac; Homo sapiens; ChIP-Seq
Frozen tissue (20-30mg for histone mark ChIP and 50-80mg for transcription factor ChIP) was pulverized using the CryoPREP dry impactor system (Covaris). The tissue was then fixed using 1% formaldehyde (Thermo fisher) in PBS for 18 minutes either at 37 degrees Celsius (histone mark ChIP) or at room temperature (transcription factor ChIP) and was quenched with 125 mM glycine. Chromatin was lysed in ice-cold lysis buffer (50mM Tris, 10mM EDTA, 1% SDS with protease inhibitor for histone mark ChIP; 0.1% SDS, 0.5% sodium deoxycholate and 1% NP-40 with protease inhibitor for transcription factor ChIP) and was sheared to 300-800 bp using the Covaris E220 sonicator (105 watt peak incident power, 5% duty cycle, 200 cycles/burst for 10 minutes for histone mark ChIP; 140 watt peak incident power, 5% duty cycle, 200 cycles/burst for 20 minutes for transcription factor ChIP). Five volumes of dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris HCl pH 8.1) were added to chromatin for histone mark ChIP. The sample was then incubated with antibodies (H3K27ac, Diagenode, C15410196; H3K27me3, Cell Signaling 9733S; H3K4me3, Diagenode C15410003 premium; FOXA1, ab23738, Abcam) coupled with protein A and protein G beads (Life Technologies) at 4 degrees Celsius overnight. The chromatin was washed with RIPA wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) for 10 minutes six times and rinsed with TE buffer (pH 8.0) once. DNA was purified using MinElute column followed by incubation in the de-crosslinking buffer (1% SDS, 0.1M NaHCO3 with Proteinase K and RNase A) at 65 degrees Celsius.  DNA sequencing libraries were prepared using the ThruPLEX-FD Prep kit (Rubicon Genomics). Libraries were sequenced using 150-base paired end reads on an Illumina platform (Novogene).
GEO Accession
GSM4928575