GSM1820964
GSM1820964: Input_293Trex_2hB4Nflag_Oct2014; Homo sapiens; ChIP-Seq
Formaldehyde cross-linked chromatin for ChIP-seq experiments with antibodies against proteins and histone modifications were prepared from 1.5 × 108 cells of each NMC cell line and 293TRex-Flag-BRD4NUT-HA as described (Alekseyenko et al. 2015). Chromatin from 0.8g of 1015 patient tissue ((Grayson et al. 2014) was prepared as follows: frozen tissue was ground to a powder with a ceramic mortar and pestle chilled with liquid N2. The powder was immediately transferred into a 50 ml Falcon tube filled with 9mL of NEB buffer (10% sucrose, 20 mM HEPES at pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton, 0.1 mM PMSF). The mixture was vortexed for 15sec to brake-up the clumps, and immediately after, 45 mL of PBS and 0.5mL of 37% formaldehyde were added to the tube. The resulting mixture was incubated for 25 min at 25°C on an orbital shaker platform with gentle mixing (150 rpm). Washing and chromatin shearing steps were performed as described (Alekseyenko et al. 2015). Affinity purification was performed as described (Kharchenko et al. 2011). Input and IP DNA recovery and ChIP-seq library preparation were performed as described (Alekseyenko et al. 2015). The following antibodies against proteins and histone modifications were used for ChIP-seq experiments: anti-H3K27ac (10 ul per IP, Active Motif, catalog no. 39133); anti-H3K9ac (15 ul per IP, Abcam, catalog no. ab12179); anti-H3K14ac (2 ul per IP, Abcam , catalog no. ab52946), anti-H3K36me3 (3 ul per IP, Abcam , catalog no. ab9050) anti-P300 (10 ul, Bethyl, catalog no. A300-358A); anti-BRD4-SL (against short and long isoforms of BRD4, 5 ul per IP, Bethyl, catalog no. A301-985A), anti-BRD4-L (against long isoforms of BRD4, 15 ul per IP, Active Motif, catalog no. 39909); anti-NUT (15 ul per IP; Cell Signaling, catalog no. 3625). ChIP-seq library preparation were performed as described (Alekseyenko et al. 2015).
GEO
GSM1820964
GEO Accession
GSM1820964