title: WL314
organism: Mus musculus
description: Protocols: Samples were prepared as previously described (doi:10.1016/j.ymeth.2009.03.001). Liver material was crosslinked in 1% formaldehyde for 20 minutes followed by a 10 minute incubation in 500 mM glycine buffer to neutralize the formaldehyde. Samples were rinsed with PBS and frozen at -80 Celsius until use. Chromatin immunoprecipitation was performed as previously described (doi:10.1016/j.ymeth.2009.03.001). Chromatin from a mass equivalent of at least 1/4 mouse liver was used for each ChIP experiment. Crosslinked tissues were dounce homogenized into a single cell suspension and rinsed with ice-cold PBS. Each pellet of crosslinked material was resuspended in 10 ml of LB1 (50 mM Hepes-KOH, pH 7.5; 140 mM NaCl; 1mM EDTA; 10% Glycerol; 0.5% NP-40 or Igepal CA-630; 0.25% Triton X-100), rocked at 4 degrees C for 10 minutes, and pelleted by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Pellets were then resuspended in 10 ml of LB2 (10 mM Tris-HCL, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.5 mM EGTA), rocked gently at 4 degrees C for 5 minutes, and pelleted again by centrifugation at 2,000 x rcf for 4 minutes at 4 degrees C. Finally each pellet was resuspended in 3 ml of LB3 (10 mM Tris-HCl, pH 8; 100 mM NaCl; 1 mM EDTA; 0.5 mM EGTA; 0.1% Na-Deoxycholate; 0.5% N-lauroylsarcosine). Chromatin from the nuclei fraction was fragmented to an average length of 300 bp by sonication. Sonicated lysates were treated with 150uL of 20% triton X-100, and sonication debris was removed by centrifugation at 20,000 x rcf for 10 minutes at 4 degrees C. 150uL of whole cell extract (WCE) was taken as the input control. 100uL of magnetic protein-G Dynabeads (LifeTechnologies, cat. no. 10004D) was washed three times and pre-blocked with 0.5% BSA (Sigma, cat. no. A8412) w/v in PBS. 5-10 ug of antibody was incubated with pre-blocked Dynabeads for at least 6 hours, rotating at 4 degrees C, then washed three times with blocking solution to remove excess unbound antibody. Antibodies used were: Rad21 (Abcam, ab992), Top2b (Santa Cruz Biotech, sc13059), H3K4me3 (Abcam, ab8580), H3K36me3 (clone 13C9 from Hitoshi Kimura), Ctcf (Millipore, mp07-729), H3K4me2 (Millipore, mp07-030). Lysates were added to protein-G bound antibodies and incubated overnight at 4 degrees C. ChIP-exo was performed as previously described (doi: 10.1093/nar/gku846). Livers from wildtype C57BL6/J male mice (~3 months old) were obtained post mortem. Approximately 1/3 of each liver was utilized per ChIP-exo experiment. 7 ug of antibody against the Top2b (Santa Cruz Biotech, sc13059), Ctcf (Millipore, mp07-729), and Rad21 (Abcam, ab992) were used for each biological replicate. Individual livers were cross-linked in 1% formaldehyde and chromatin immunoprecipitation (ChIP) was performed as previously described up until and including the RIPA buffer washes (step 38 from (doi:10.1016/j.ymeth.2009.03.001)). The ChIP-exo portion of the protocol was performed as previously described (doi: 10.1016/j.cell.2011.11.013) with modifications making the assay compatible with the Illumina sequencing platform. Briefly, while still on the magnetic beads (Invitrogen, Dynabeads), the immunoprecipitated DNA was end-repaired (NebNext End Repair Module, New England Biolabs (NEB)). The P7 adapter was designed based on the adapter sequence provided by NEB: 5'-Phos-TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3' and 5'-AGATCGGAAGAGCACACGTCTGAACTCC-3'. After ligation, the DNA was nick-repaired with phi29 polymerase, and digested by lambda and RecJf exonucleases (NEB). DNA samples were eluted from the beads by performing reverse crosslinking overnight at 65 degrees C, followed by RNaseA (Ambion) and ProteinaseK (Invitrogen) treatments. DNA was extracted using phenol-chloroform-isoamyl protocol and ethanol precipitation. DNA was denatured at 95 degrees C, 3' ends were primer-extended with a P7 fill-in primer (5'-TGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'), and phi29 polymerase. A second adaptor ligation to only lambda exonuclease-digested ends of double stranded DNA was performed using 15 pmol of P5 adapter (sequence provided by NEB: 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTG-3' and 5'-TCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'). The resulting DNA samples were PCR-amplified using multiplexing index primers (NEBNext DNA Library Prep Master Mix Set for Illumina). Libraries were size selected from 180-300bp using a 2% agarose dye-free automated size selection cassettes from Pippin Prep (Sage Sciences, cat. no. CDF2010), and quantified with 2100 Bioanalyzer (Agilent).