GSM1704278: R1b_fS248A_long; Mus musculus; ChIP-Seq
Crosslinking and Sonication: The cells from ∼80% confluent 10 cm dishes were crosslinked with 1% formaldehyde for 10 minutes at room temperature. After quenching with 125 mM glycine for 5 min, the cells were washed twice with ice cold 1xPBS. Cell pellets were resuspended in 1ml lysis buffer 1 (50 mM HEPES-KOH (pH 7.6), 140 mM NaCl, 1 mM EDTA, 10%(v/v) Glycerol, 0.5% NP-40, 0.25% Triton X-100, 1X complete protease inhibitor cocktail (PIC) Roche, 11697498001) for 10 min at 4 oC, followed by spin at 1,350 x g for 5 min at 4oC. Discard the supernatant and resuspend the pellet in 1ml of lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1X PIC) for 10 min at 4oC, followed by spin at 1,350 x g for 5 min at 4oC. Finally, resuspend the cell pellet in 700 µl of lysis buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Sodium deoxycholate, 0.5% N-Lauroylsarcosine, 1X PIC) and sonicate to desired length using Bioruptor (UCD-200; Diagenode, Delville, NJ, USA) for 30 min [10 minutes x 3 times (each cycle consists of 30s on, 30s off) at high settings]. Clarify the lysates by centrifugation at 10,000 rpm in a microcentrifuge for 10min at 4oC. Transfer the supernatant to a new tube and store at -80oC or use it immediately for ChIP. We had two sets of chromatin due to differences in fragment size distribution generated after sonication (long and short) and we used both for library preparation. Long chromatin library was generated from chromatin that still had high molecular weight fragments (1-2Kb-500bp) even after sonication. Short chromatin library was generated using sheared chromatin that had majority of fragments under small molecular weight range (500-200 bp). Chromatin Immunoprecipitation using FLAG antibody: 50μl Protein G Magnetic beads (New England Biolabs) were washed twice with PBS with 5 mg/ml BSA and 10 μg of anti-Flag M2 antibody (Sigma, F1805) coupled in 500 μl PBS with 5 mg/ml BSA overnight at 4°C. Immunoprecipitation was performed with antibody-coupled beads and sonicated supernatants in ChIP buffer (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) overnight at 4°C. The magnetic beads were washed twice with ChIP buffer, once with ChIP buffer including 500 mM NaCl, four times with RIPA buffer (10 mM Tris-HCl pH8.0, 0.25M LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate), and once with TE buffer (pH 8.0). Chromatin was eluted twice from washed beads by adding elution buffer (20 mM Tris-HCl pH8.0, 100 mM NaCl, 20 mM EDTA, 1% SDS) and incubating for 15 min at 65°C. The eluted DNA and the input samples were then reverse crosslinked at 65oC for overnight, followed by RNase A(0.2 mg/ml) digestion at 37oC for 2h and Proteinase K (0.2 µg/µl) (NEB, P8102S) digestion at 55oC for 1h. The ChIP and input DNA were recovered by phenol-chloroform extraction and ethanol precipitation. ChIP samples were end-repaired, A-tailed and adaptor ligated using barcode adaptors. 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 Qiagen mini elute columns was performed following each enzyme reactions. The adaptor ligated material was then PCR amplified with Phusion polymerase using 16 cycles of PCR before size selection of 200-350 bp fragments on a 2% agarose gel. The ChIP-seq library samples were purified using Qiagen gel extraction kit, and fragment size distribution was determined on Agilent Bioanalyzer using High Sensitivity DNA chip. Library concentrations were measured using KAPA Library Quantification Kits (Kapa Biosystems, Inc., Woburn, MA) and equal amounts of indexed libraries were pooled and sequenced on the Illumina HiSeq 2000. Sequencing was performed at Center for Advanced Technology, genomics core facility at University of California, San Francisco.
GEO Accession
GSM1704278