GSM3293962: H3 control replicate 2; Homo sapiens; ChIP-Seq
Samples for ChIP Sequencing were prepared using SimpleChIP Kit # 9003 from Cell Signaling Technology without modifications. Cells HT1080, tail fibroblasts from SSRP1 KO mice were grown to 80% confluence, fixed with 1% formaldehyde for 10 min and  protocol for this kit was followed: chromatin was digested with Micrococcal Nuclease into 150-900 bp DNA/protein fragments, antibodies specific to H3 ( provided by kit) and to H3 acetyl K27 ( ab4729, Abcam) were added and the complex co-precipitated and was captured by protein G magnetic beads. Cross-links were reversed, and DNA was purified for sequencing. 20ng of Chromatin-immunoprecipitated (ChIP) DNA is used to generate a library for Next generation sequencing using the ThruPLEX DNA seq kit (Rubicon Genomics, Inc.) as per manufacturer's instructions.  ThruPLEX uses stem-loop adapters to construct high quality libraries in a fast and efficient workflow. In the first step, Template Preparation, the DNA is repaired and yields molecules with blunt ends. Next, stem-loop adaptors with blocked 5' ends are ligated with high efficiency to the 5' end of the genomic DNA, leaving a nick at the 3' end.  In the final step, the 3' ends of the genomic DNA are extended to complete library synthesis and Illumina-compatible indexes are added through a high-fidelity amplification. The library is quantitated using quantitative PCR (KAPA Biosystems), normalized, and pooled in an equimolar fashion.  Pooled libraries are loaded to an individual lane of a HiSeq High Output v3 flow cell, using an Illumina cBot, for 2 x 75 PE and 75bp single end  sequencing on a NextSeq sequencer according to the manufacturer's recommended protocol (Illumina Inc.).
GEO Accession
GSM3293962