alias: GSM4544341
title: GSM4544341: H3K4me3_kidney_wt_rep2; Mus musculus; ChIP-Seq
library_construction_protocol: Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. ChIP-nexus libraries were performed as described (He et al., 2015), with some modifications. Briefly, The chromatin-enriched magnetic beads were then washed with buffers, repaired with the end-repair enzyme mixture (NEB) and dA-tailed with Klenow exo- (NEB) in NEB buffer 2 containing 0.2 mM dATP. After ligating the Nexus adaptors to DNA with 2x Blunt/TA Ligase Master Mix (NEB, M0367S) for 1h at 25 ℃, dealt the mixtures with Klenow exo- and followed by ligation with T4 DNA polymerase (NEB). The blunt-ended DNAs were then treated with Lambda Exonuclease in its buffer (NEB). Subsequently, samples were then digested by RecJf exonuclease (NEB) and washed three times with the RIPA buffer. DNA was then eluted, reverse-crosslinked, purified with phenol: chloroform to get ssDNA.  The ssDNA was circularized with CircLigase (Epicentre), annealed with the Cut_oligo in FastDigest buffer (Thermo), and then digested with FastDigest BamHI (Thermo),  precipitated using ethanol and sodium acetate, resuspended with 20 μl of water.DNAs were amplified by Q5 polymerase (NEB) with Illumina universal primer and index primers.  The PCR products with the sizes of 150 bp to 300bp were extracted from 2% agarose gel using MinElute Gel Extraction Kit (Qiagen).ChIP-Seq libraries were prepared according to the manual of VAHTSTM Universal DNA Library Prep Kit for Illumina® V2 (ND606). Briefly, DNA was end-repaired using End Prep Mix 3, ligated with index-contained adapters using Rapid DNA ligase in Rapid Ligation buffer, amplificated using PCR Primers and Amplification Mix for libraries. The library fragments of ~350 bp (insert plus adaptor and PCR primer sequences) were isolated  with Agencourt® AMPure® XP (Beckman Coulter®). Library DNAs were sequenced on an Illumina HiSeq platform
identifiers.namespace: GEO
identifiers.id: GSM4544341