title: GSM2309225: NCCIT_Input; Homo sapiens; ChIP-Seq
library_construction_protocol: Mononucleosomes were isolated by MNase digestion of cells, followed by formaldehyde fixing and fractionating the concentrated nucleosome mixture it on a 5-25% sucrose gradient, to purify the mononucleosome fraction away from dinucleosomes. Alternate "Quick protocol" involved controlled detergent mediated treatment of NCCIT cells and digestion with a fixed concentration of MNase that allows release of mononucleosomes into the supernatant that are crosslinked and used for ChIP. Modified Sequential ChIP involved crosslinking of the 1st Antibody to beads by DSG followed by elution with SDS/65degC and the elute used for sequential pulldown using the 2nd Antibody. DNA was extracted using Qiagen PCR purification preps from the decrosslinked eluate of sequential ChIP and was used either for western blotting or PCR. 10ng of seq ChIP DNA was amplified by WGA using Illumina kits for Library preparation. Libraries were constructed according to the protocol provided by Illumina’s TruSeq DNA Sample Preparation v2 Kit.  Library prep begins with the End Repair and dA tailing followed by Illumina indexed sequencing adaptor ligation that are complementary for hybridization on the flow cell.  PCR amplification was used to amplify the DNA fragments for 15 cycles and assessment of the yield and size distribution of the amplified library was performed on the Agilent 2100 Bioanalyzer using the High Sensitivity Chip.  Quantitation of the libraries was performed by qPCR using the KAPA SYBER FAST qPCR Kit supplied by Kapa Biosystems according to manufactures’ protocol.  Multiplex libraries were pooled to a final concentration of 2nM and cluster generation completed on Illumina’s cBot using the TruSeq SR Cluster Kit v3.  Single read 50bp sequencing was performed on the Illumina HiSeq2000 with the TruSeq SBS Kit v3 (50cycles).