GSM1302274: Pooled Input chromatin; Rattus norvegicus; ChIP-Seq
Chromatin was prepared from approximately 25 million siTCF7L2 or scramble electroporated cells by shearing with MNase using the SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) to fragments ranging between 150 bp and 900 bp. Each aliquot was incubated overnight with either 5 ul of the anti-TCF7L2 antibody or 1 µl of the anti-HNF4α antibody. A sample consisting of 2% of the total input chromatin was removed from each and served as the sequencing control. Immunoprecipitated material was purified as described by the manufacturer (Cell Signaling Technology), except that replicate immunoprecipitations were pooled at the final Qiagen DNA purification step. Each sequenced sample represents a pool of 2 independent immunoprecipitations, each consisting of between 10 and 20 ug of chromatin. ChIP-Seq libraries were generated using a combination of Illumina TruSeq adapters/indexes and a Kapa Library Preparation Kit (Kapa Biosystems, Woburn, MA, USA; cat # KK8200) with the following modifications. End-repair was performed on approximately 5 – 10 ng of ChIP sample. Qiagen Minelute purifications (Qiagen, Valencia, CA, USA) were utilized in between each step of adenylation and adapter ligation. Adapter ligation was carried out using stock Illumina adapters and Kapa T4 ligase at 30°C for 10 minutes, followed by a single DNA clean-up using homemade SPRI beads. The adapted material was minimally amplified for 5 cycles, using Illumina PCR primer cocktail and Kapa HiFi HotStart Polymerase ReadyMix (cat # KK2601), to convert y-shaped adapters to double-stranded DNA for more reliable electrophoresis, and then size selected on a 2% agarose gel before a final PCR amplification of 8 – 12 cycles and SPRI bead clean-up. Final libraries were checked for size using Bioanalyzer HS DNA or DNA 1000 chips.
GEO Accession
GSM1302274