title: do178
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 libraries were made as previously described protocol (doi:10.1016/j.ymeth.2009.03.001). 50 µl of cell lysate from each sonication was removed as the input control DNA that was processed. Libraries were prepared following the instructions of Illumina (Sample preparation for genomic DNA - version 2.2) with the following modifications: The ChIP-enriched DNA and input DNA were not further fragmented. After end-repair and addition of an 'A' base to the 3' ends, the adapters were ligated to the ends of the DNA fragments using 2 µl of forty-fold diluted 'Adapter oligo mix'' in a total reaction volume of 25 µl. Between these steps, the DNA was purified using the DNA Clean-and-Concentrator-5 kit (Zymo Research). Subsequently, the DNA was amplified by 18 cycles of PCR, purified with QIAquick PCR purification Kit, and eluted with 33.5 µl of 10 mM Tris buffer at pH7.0. The PCR product was sized selected on 2% agarose gel and a gel slice containing the 200-300 bp fragments was excised.