GSM1944105: KMT2D ChIP-Exo 3; Mus musculus; ChIP-Seq
ChIP-Exo Library Prep: All adapter and primer sequences were obtained from Serandour et al., Genome Biology, 2013.  Briefly, immunoprecipitation was performed using a mixture of Protein G magnetic beads, anti-KMT2D antibody, and crosslinked KMT2D-DNA complexes.  Beads were collected using a magnet and all supernatant (containing unbound material) was removed.  Several enzymatic reactions were then performed while the immunoprecipitated KMT2D-DNA complexes were still bound to the beads; a number of washes preceded each step.  First, complexes were treated with T4 DNA polymerase, E. coli Pol I large (Klenow) fragment, and T4 polynucleotide kinase to ensure all dsDNA fragments were blunt-ended with 5' phosphates.  Next, P7 adapter was ligated to each fragment using T4 DNA ligase.  Following ligation, nicks were repaired using Phi29 polymerase.  KMT2D-DNA complexes were then digested with Lambda exonuclease, which acts only in the 5' to 3' direction.  This step leaves behind single-stranded DNA except where crosslinked protein protects the DNA from digestion.  Following this first digestion, a second digestion was performed using RecJf, a fusion protein of RecJ and maltose binding protein.  This enzyme specifically digests single-stranded DNA in the 5' to 3' direction.  Finally, immunoprecipitated KMT2D-DNA complexes were eluted and crosslinks were reversed overnight (65C, 0.54M NaCl).  DNA was isolated from each sample.  Complimentary strand synthesis was completed by denaturing the DNA and then treating with Phi29 polymerase following the addition of a primer complimentary to the P7 adapter sequence.  DNA was isolated and P5 adapter was ligated to each strand using T4 DNA ligase.  DNA was again isolated and amplified using a pair of universal and barcoded primers.  Amplified fragments between 200 and 400bp were isolated from an agarose gel and purified.  This DNA was then clustered on an Illumina flow cell and sequenced on an Illumina HiSeq 4000. Both WT and mutant hearts were thawed on ice and pooled into 3 replicates of 30 hearts each. Samples were crosslinked with 1% formaldehyde, lysed with cell lysis and nuclei lysis buffer, and sonicated to shear chromatin into 200-600 bp fragments. Each replicate was further split into input and 3 samples. Chromatin immunoprecipitation was performed with 3 ug of anti-H3K4me1/2/3 antibody (Abcam) overnight with Protein G Dynabead (Life Technologies). After washing, DNA was eluted, reverse-crosslinked and precipitated with AmPure XP magnetic beads (Beckman Coulter).
GEO Accession
GSM1944105