title: GSM7136977: ChIP-seq SPT5 shCTR9 EtOH; Mus musculus; ChIP-Seq
library_construction_protocol: ChIP-seqCells were treated with 1% formaldehyde for 5 min at 37°C. After cell lysis, nuclei were re-suspended in RIPA buffer (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% NP40, 1% deoxycholic acid (DOC), 0.1% SDS, 1 mM EDTA) and DNA was fragmented to a size <300 bp using sonification. Chromatin was immunoprecipitated with 15 µg of corresponding antibody for 6 hours. Chromatin was eluted with 1% SDS and crosslinking was reverted overnight. For purification, chloroform/phenol extraction was used. CUT&RUN: cells were harvested using Accutase (Sigma) for cell detachement. Cells were counted and washed in wash buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine). After washing, cells were coupled to ConA-coated magnetic beads (Polysciences Europe) and subsequently permeabilized and followed by addition of primary antibody in antibody binding buffer (wash buffer, 0.05% digitonin, 2 mM EDTA) over night at 4 °C. Cells were wased in Dig-Wash buffer (Wash buffer, 0.05% digitonin) and MNase (700ng/ml) was added to each sample for 1 hr at 4 °C. After washing with Dig-wash buffer and Low-salt Rinse buffer (20 mM HEPES pH 7.5, 0.5 mM Spermidinge, 0.05% digitonin) incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.05% digitoin) was added for 30 min at 4°C. STOP buffer (170 mM NaCL, 20 mM EGTA, 0.05% digitonin, 20 ug/ml RMAse A, 25 ug/ml Glycogen was added to stop the MNase digestion. DNA fragments were released at 37 °C for 30 min and proteins digested for 1 h at 50 °C after adding 0.01 % SDS and 10 mg/ml Proteinase K followed by Phenol Chloroform extraction. Precipitated DNA was resuspended in 30 ul 0.1x TE buffer ChIP-seq Libraries were prepared according to instructions accompanying the NEBNext Ultra™ II DNA Library Prep Kit for Illumina. Purified DNA was end-repaired, A-tailed, ligated to Illumina adaptors, size-selected (150 bp) and eluted. DNA fragments were amplified by 11 to 16 cycles of PCR and library size was tested with the Fragment Analyzer (Advanced Analytical). The amount of library DNA was quantified using a picogreen assay. CUT&RUN: NEBnext Ultra II DNA Library Prep Kit (New England Biolabs) was used. Pre-PCR samples were purified using AmpureXP beads with a ratio of 1.75x. Then half of the eluted material was used with 12-16 PCR cycles. The libraries were cleaned using Agentcourt AMPure XP Beads (ratio 1.1x, Beckman Coulter), quality, quantity and fragment size assessed on the Fragment Analyzer (Agilent) using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Agilent)