title: GSM1269902: Hdac6KD_p400ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Cells from ~80 % confluent 10 cm dishes were crosslinked by adding fixation solution (1% formaldehyde, 0.1M NaCl, 1mM EDTA, 50mM HEPES⋅KOH pH 7.6) for 10 minutes at room temperature. Crosslinking was quenched with 125mM Glycine for 5 minutes. Cells were washed twice with cold PBS containing protease inhibitors (Roche), and pelleted at 1000g for 5 minutes at 4°C. Cell pellets were either flash frozen in liquid nitrogen and stored at -80°C or immediately sonicated. Pellets were resuspended in Lysis buffer 1 (50mM HEPES⋅KOH pH 7.6, 140mM NaCl, 1mM EDTA, 10% (v/v) Glycerol, 0.5% NP-40, 0.25% Triton X-100) including protease inhibitors and incubated for 10 minutes at 4°C. After centrifugation at 1350g for 5 minutes, pellets were resuspended in Lysis buffer 2 (10mM Tris-HCl pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA) containing protease inhibitors and incubated for another 10 minutes at 4°C. Pellets were collected after centrifugation at 1350g for 5 minutes and resuspended in Lysis buffer 2 for sonication. Samples were transferred into 15 ml tubes (Falcon) and sonicated in a Bioruptor set to high for 2 cycles (10 minutes for one cycle with 30 sec on/ 30 sec off). The supernatants were collected after a 13000 rpm spin for 10 minutes at 4°C. 50μl Protein G Magnetic beads (NEB) were washed twice with PBS with 5mg/ml BSA and 10μg of anti-Flag M2 antibody (Sigma) coupled in 500μl PBS with 5mg/ml BSA overnight at 4°C. Immunoprecipitation was performed with antibody-coupled beads and sonicated supernatants in ChIP buffer (20mM Tris-HCl pH8.0, 150mM NaCl, 2mM EDTA, 1% Triton X-100) overnight at 4°C. Magnetic beads were washed twice with ChIP buffer, once with ChIP buffer including 500mM NaCl, 4 times with RIPA buffer (10mM Tris-HCl pH8.0, 0.25M LiCl, 1mM EDTA, 0.5% NP-40, 0.5% Na⋅Deoxycholate), and once with TE buffer (pH 8.0). Chromatin was eluted twice from washed beads by adding elution buffer (20mM Tris-HCl pH8.0, 100mM NaCl, 20mM EDTA, 1% SDS) and incubating for 15 minutes at 65°C. The crosslinking was reversed at 65°C for 6hr and RNase A (Sigma) was added for 1hr at 37°C followed by proteinase K (Ambion) treatment overnight at 50°C. ChIP-enriched DNA was purified using Phenol/Chloroform/Isoamyl alcohol extractions in phase-lock tubes. Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (exo minus) and dATP to yield a protruding 3- 'A' base for ligation of barcoded adapters which have a single 'T' base overhang at the 3’ end. DNA purification on Zymo Research PCR purification columns (Zymo, Irvine, CA) was performed following each enzyme reaction. The adaptor-ligated material was then PCR amplified with Phusion polymerase using 16 cycles of PCR before size selection of 200-300 bp fragments on a 2% agarose gel. Libraries with different barcodes were pooled together and single-end sequencing (50 bp) was performed on an Illumina HiSeq2000.
attributes.tag: GEO Accession
attributes.value: GSM1269902