GSM1572784: Sample_H3K4me3_Index_2_lane6; Homo sapiens; ChIP-Seq
Chromatin was then subjected to phenol:chloroform extraction followed by purification using a PCR purification kit (Qiagen, Germantown, Maryland). Quantitative real time PCR (qRT-PCR) on positive housekeeping genes was performed on both input and eluted chromatin, to validate the ChIP efficacy. Libraries compatible with Illumina TruSeq adapter sequencing (Illumina Inc, San Diego, CA) were made as follows: 10 ng of either immunoprecipitated or input DNA were end-repaired using 3 units of T4 DNA polymerase, 1 unit of Klenow DNA polymerase and 10 units of T4 DNA polymerase with 30 minutes incubation at 20ºC;  A-tailing was performed with 5 units of Klenow fragment and 10mM dATP (New England Biolabs, Ipswich, MA) for 30 minutes at 37ºC. 2 ul of 1uM of annealed Illumina TruSeq adapters (Integrated DNA Technologies, Coralville, IA) were used in an overnight ligation at 4ºC with 2000 units of T4 DNA Ligase. To account for the migration pattern of Y-forked Illumina adaptors ligated products from 250-350bp were size selected in a 1% agarose gel. After purification, the PCR reaction was carried out with 300uM dNTP, 200uM of primers and 1 unit of Phusion High-Fidelity DNA Polymerase (Thermo Scientific, Rockford, IL). Initial denaturation of 94ºCx5 min, was followed by18 cycles of 94ºCx20secs, 60ºCx30secs, 72ºCx30secs, with a final extension/elongation step of 72ºCx5min. PCR product was cleaned by the use of SPRI beads as per manufactor’s recommendation [Beckman Coulter, Indianapolis, IN]. Final product was resuspended in 20 ul of TrisEDTA. Final yields were quantified in a Qubit 2.0 Fluorometer (Life Technologies, Grand Island, NY) and quality of the library was assessed by running on a DNA1000 Bioanalyzer chip (Agilent Technologies, Santa Clara, CA). Libraries were normalized to 2nM and loaded on an Illumina HiSeq 2500 at 6pM, per manufacturer’s recommended protocol for 50bp single-read runs.
GEO
GSM1572784