GSM6938942: CnR-EXOSC5-DOX-EtOH; Homo sapiens; OTHER
For Cut&Run sequencing, cells were harvested with Accutase, washed and then coupled to ConA-coated magentic beads (Polysciences Europe). Cells were then permeabilized and incubated with the primary antibody (EXOSC5 #NBP2-14952; 1:100 diluted; Novus Biologicals) in antibody binding buffer over night at 4°C. Cells were then washed in the presence of digitonin and 700 ng/ml MNase was added per sample for 1 hour at 4°C. For Cut&Run sequencing samples, the MNase treated cells were washed with low-salt rinse buffer. Incubation buffer, containing CaCl2 and digitonin, was added for 30 min at 4°C. The Mnase activity was stoped in the presence of 20 µg/ml Rnase A. DNA fragments were released at 37°C for 30 min and the decrosslinking was done for 1 hour at 50°C by adding 0.01% SDS and 10 mg/ml Proteinase K. DNA was extracted wit phenol-chloroform and resuspended in TE buffer. For ChIP-seq and ChIP-Rx sequencing, cells were fixed for 5 min at RT with 1% formaldehyde and harvested in cold PBS contianing protease and phosphatase inhibitors. For ChIP-Rx samples, murine NHO2A or NIH3T3 cells were added at a 1:10 cell ratio during cell lysis to serve as an exogenous control (spike-in). Cell lysis was done for 20 min in PIPES buffer containing 85 mM KCl and 0.5% NP-40, followed by collection of nuclei by centrifugation. For ChIP-seq and ChIP-Rx sequencing, the crosslinked chromatin was extracted from the nuclei in TRIS buffer with 150 mMCaCl, 1 mM EDTA and 1% NP-40, 1% sodium deoxycholate and 0.1% SDS. The chromatin was fragmented using the Covaris Focused Ultrasonicator M220 for 50 min. Dynabeads Protein A and Protein G were pre-incubated overnight with 10-15 µg antibody in the presence of 5 mg/ml BSA. For ChIP-seq and ChIP-Rx sequencing, chromatin was added to the pre-incubated beads for at least 6h at 4°C with rotation. Beads were then washed with different TRIS buffers containing distinct salt-concentrations, EDTA and detergents, followed by wahsing with only TE-buffer. Chromatin was eluted with 100mM NaHCO3 and 1% SDS for 15 min. Protein and RNA were digested with proteinase K and RNase A. DNA was isolated by phenol-chloroform extraction followed by an ethanol precipitation. DNA was quantified using the Quant-IT PicoGreen dsDNA assay. The DNA libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. Pre-PCR samples were purified and/or size-selected using AMPureXP beads. The libraries were PCR-amplified with the NEBNext Multiplex Oligos for Illumina Kit and again purified using Agencourt AMPure XP beads (Beckman Coulter). Library quality and fragment size distribution was analyzed on a Fragment Analyzer (Advanced Analytical). Sequencing was performed in single-read or paired-end mode on a NextSeq500 or NextSeq2000 platform (Illumina). ChIP, ChIP-Rx and CUT&RUN followed by sequencing.