title: WL121
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 DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligating Illumina adaptors using NEBNext DNA library preparation kit (New England Biolabs, cat. no. E6040L). ChIP DNA and 220ng of WCE were end repaired for 30 minutes at room temperature, and purified using either DNA Clean and Concentrator kit (Zymogen, cat. no. D4014) or PCR column purification kit (Qiagen, cat. no. 28106) following manufacturers protocols. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37 degrees C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T-overhang and a proprietary uracil hairpin. USER enzyme was used to cleave the uracil hairpin, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primers (New England Biolabs, cat. no. E7335L) in order to multiplex the libraries for sequencing. PCR-amplified DNA was purified using PCR column purification and eluted in 20uL of elution buffer for gel extraction, or 30uL of TE for the Pippin Prep size selection. Size selection was performed in one of two ways: (i) 200-300bp region was gel purified by casting a 2% ultra-pure ChIP-grade agarose gel and MinElute Gel Extraction Kit, or (ii) samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassettes from Pippin Prep (Sage Sciences, cat. no. CDF2010).