GSM5392262: CAGE-seq.GSC_316_DMSO_R1; Homo sapiens; RNA-Seq
[Epigenomic profiling]  GSC316 cells were treated with 0.5% formaldehyde for 6 min at room temperature to crosslink histone to DNA, which was followed by the addition of 1.25 M glycine to quench the crosslinking reaction. Cells were subsequently washed by ice-cold PBS with BSA (5 mg/ml), only ice-cold PBS, and PBS with protease inhibitors. After aspiration of supernatant, cell pellets were flash-frozen in liquid nitrogen. Flash-frozen cell pellets were stored at -80 °C. Cell pellets were lysed with lysis buffer (50 mM Tris-HCl pH 8.1 containing 1% SDS, 10 mM EDTA, and protease inhibitors-4693116001, Roche) by incubation on ice for 10 min, followed to sonication by Covaris (Duty Cycle: 5%, Cycles per Burst: 200, Volume 130 μl) for 8 min at low cell chromatin shearing mode. Sizes of sheared chromatin were examined by Agilent Bioanalyzer. The sheared chromatin was diluted in the dilution buffer (20 mM Tris-HCl pH 8.1 containing 150 mM NaCl, 1% Triton, and 2 mM EDTA). A fraction of sheared chromatin was prepared for input control by treating with RNase A and Proteinase K and heated for de-crosslinking. After clearing, the sheared chromatin was incubated for 10 to 12 hrs at 4 °C with antibody bounded magnetic beads (40 μl of DynabeadsTM sheep anti-mouse IgG, Cat. no. 11201D or sheep anti-rabbit IgG, Cat. no. 11203D for each ChIP. A total of 2.5 to 10 μg of each antibody per 2.5 – 8 × 106 cells was used. Specifically, anti-H4K5acK8ac (3 μg for 4 × 106 cells), anti-H3K4me3 (3 μg for 3 × 106 cells), anti-H3K27ac (2.5 μg for 3 × 106 cells), anti-H3K4me1 (4 μg for 4 × 106 cells), and anti-BRD4 (10 μg for 8 × 106 cells) antibodies were used for each ChIP. The antibody-bead conjugated chromatin was subsequently washed two times with low-salt wash buffer (20 mM Tris-HCl pH 8.1 containing 150 mM NaCl, 0.1% SDS, 2 mM EDTA, and 1% Triton X-100), two times with high-salt wash buffer (20 mM Tris-HCl pH 8.1 containing 500 mM NaCl, 0.1% SDS, 2 mM EDTA, and 1% Triton X-100), two times with LiCl wash buffer (10 mM Tris-HCl pH 8.1 containing 0.25 M LiCl, 1% IGEPAL-CA630, 1% deoxycholic acid, and 1 mM EDTA) and twice with Tris-EDTA wash buffer (10 mM Tris-HCl pH 8.1 containing 1 mM EDTA). For BRD4-ChIP, single washes for each of low-salt, high-salt, LiCl wash buffer and twice for Tris-EDTA wash buffer were used. The chromatin was eluted in elution buffer (100 mM NaHCO3 containing 1% SDS) and de-crosslinked at 65 °C for 5 to 6 hrs. RNA and protein were digested using 1 μl of RNase A and 1.5 μl of Proteinase K (20 mg/ml). The DNA was then isolated using Qiagen MinElute column purification for U87 and C13NJ cell lines. The Agencourt AMPure XP (A63880, Beckman Coulter) was used for DNA purification in GSC316. DNA was then subjected to qPCR with locus-specific primers. The ChIP-qPCR results were normalized to the input DNA. [Transcriptomic profiling]  RNA samples from cells (DMSO-treated, JQ1-treated, and untreated) of GSC316, U87, and C13NJ were subjected for cap-analysis of gene expression (CAGE) transcriptome profiling. RNA was isolated from the nuclear fractions of three biological replicates of each treatment group. [ChIP-seq]  For ChIP-seq library preparation, Mondrian (2 ng of ChIP DNA and 11 PCR-cycles) was used and each ChIP-seq library was subjected to 50-bp sequencing using HiSeq 2500. [CAGE-seq]  The RNA integrity value of the above nine samples was confirmed through Agilent Bioanalyzer and RNA (at least 3 μg of each sample) samples were subjected for CAGE library preparation. Each CAGE library was sequenced at least 15 million reads. Illumina HiSeq2500 High output run mode
GEO Accession
GSM5392262