GSM4980450: HEK293_VPRP_D1399Y_H3K27Ac_1; Homo sapiens; ChIP-Seq
RNA-seq was performed from duplicates for each condition. To prepare samples, cells were induced with dox for 48 hours and GFP+/BFP+ double positive cells were sorted on a BD Influx flow cytometer (BD Biosciences). WT cells containing only an sgRNA were sorted for BFP+ cells. 1 million cells were sorted for each sample and total RNA was purified using RNeasy Plus mini kit (Qiagen). H3K27ac ChIP-seq was performed from duplicates for each condition. To prepare samples, cells were induced with dox for 48 hours. To fix the cells, 1/10 volume of freshly-prepared Formaldehyde solution (11% Formaldehyde, 0.1M NaCl, 1mM EDTA, 50mM HEPES in water) was added to existing media for 15 minutes at room temperature. The fixation was stopped by adding 1/20 volume of 2.5M Glycine solution for 5 minutes at room temperature. After the glycine incubation cells were scraped from the culture surface. Cells were collected and kept on ice for the remainder of the procedure. The cells were centrifuged at 800xg for 10 minutes and then re-suspended in 10 mL chilled PBS-Igepal. Cells were centrifuged again and resuspended in 10 mL PBS-Igepal and 100ul of PMSF (100mM). Cells were centrifuged a third time and cell pellets were snap-frozen on dry ice and stored at -80C. ChIP-seq was performed by Active Motif. Total RNA quantification and qualification was done by Nanodrop for RNA purity, agarose gel electrophoresis and Agilent 2100 for RNA integrity. mRNA was purified using poly-T oligo-attached magnetic beads and fragmented. cDNA was synthesized using M-MuLV Reverse Transcriptase and DNA Polymerase I and purified using AMPure XP beads.The 3' ends of the cDNA were adenylated and a NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. AMPure XP system (Beckman Coulter, Beverly, USA) was utilized to enrich for fragments of 150-200bp length. Finally, the final library is obtained by PCR amplification and purification of PCR products by AMPure XP beads. Libraries were diluted to 1.5ng/ul (Qubit2.0) and insert size detected by Agilent 2100. Q-PCR is used to accurately quantify the library effective concentration (> 2nM). Samples were then pooled and run on Illumina sequencers. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75 nt reads, single end).