title: GSM3003987: JM08_ChIA-PET-1_ZT06_rep1; Mus musculus; ChIA-PET
library_construction_protocol: Mouse liver were collected at ZT06 (middle of the day) or ZT18 (middle of the night), rinsed in ice-cold 1X PBS and flash-frozen. Frozen livers were crushed to a fine powder in liquid nitrogen using a mortar, and crosslinked with 1% formaldehyde (JM11) or with 1. 5mM EGS and 1% formaldehyde (JM08 and JM12). Nuclei were purified, sonciated, and sonicated. Sonicated chromatin was then used to isolate Pol II -DNA complexes with an antibody. Nuclei were sonicated in 10 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% SDS, 0.2% Triton, 1X protease inhibitor cocktail. Chromatin samples from 3-7 livers were pulled together, diluted 5-fold to obtain a final concentration of ChIP buffer of 10mM Tris-Cl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail. Pol II ChIP were performed in 50 ml canonical tube with 65 µg of anti-RNA Polymerase II 8WG16 monoclonal antibody (# MMS-126R, Covance). Immunoprecipitated chromatin was washed once with TSE I buffer (10mM Tris pH 7.5, 0.1%SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl, 1mM DTT, 1 mM PMSF), once with TSE II buffer (10mM Tris pH 7.5, 0.1%SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 1mM DTT, 1mM PMSF), once with LiCl Buffer III (10mM Tris pH 7.5, 0.25M LiCl, 1% NP-40, 1% Na Deoxycholate, 1mM EDTA, 1mM DTT, 1mM PMSF), and once with TET buffer (10mM Tris pH 7.5, 1mM EDTA, 0.1% Triton). Beads were finally resuspended in 2 ml of 1X TE buffer supplemented with 1X TE. Immunoprecipitated chromatin was end-repaired, processed for ligation with biotinylated half-linkers, and 5' phosphorylated while still complexed with Pol II antibodies on magnetic beads following published protocols. Chromatin was then eluted, diluted to a final volume of 10 ml, and used for the proximity ligation step (performed for a minimum of 16 hours at 4ºC under extremely diluted conditions, i.e., < 0.2 ng DNA/μL, to favor ligation events within individual crosslinked chromatin complexes). Following the proximity ligation step, chromatin was treated with proteinase K, reverse crosslinked, and the DNA purified. ChIA-PET DNA was then digested with MmeI, immobilized on streptavidin beads, and ligated to the ChIA-PET adapters. The DNA samples were finally proceeded through nick translation and a PCR amplification step to generate the library. Libraries were ran on an agarose gel, and fragment of ~229 bp were gel-extracted, purified, and quantified.
attributes.tag: GEO Accession
attributes.value: GSM3003987