GSM2803434: AV55_CTCF_NoTx_2; Mus musculus; ChIP-Seq
Cells were fixed with 1% formaldehyde and the reaction subsequently quenched using 0.125 M glycine.  After fixation, cells were lysed to release nuclei, which were washed and re-suspended in nuclei lysis buffer before sonicating the chromatin using a Diagenode Bioruptor.  Fragmented chromatin was diluted and separated into equivalent aliquots prior to antibody application and complex retrieval. A ChIP-seq script was selected for sequencing library preparation using PrepX DNA Library kit and Apollo 324 NGS automatic library prep system (WaferGen, Fremont, CA). Overhangs in samples of double-strand DNA fragments (~5 ng) were first blunt-ended, and adenylated at the 3' ends for TA ligation to the sequencing adaptors. The ligated library was enriched by 8 cycles of PCR using index-specific primers, followed by automated AMPure XP beads (Beckman Coulte, Brea CA) purification. To check the quality and yield of the library, one µl of purified PCR library was analyzed by Bioanalyzer with DNA High Sensitivity chip. To accurately quantify the library concentration for cluster generation, the library was 1:104 diluted with dilution buffer (10 mM Tris-HCl, pH 8.0 with 0.05% Tween 20), and qPCR analyzed with NEBNext Library Quant Kit (New England Biolabs, Ipswich, MA) using ABI 9700HT real-time PCR system (Lifetech, Grand Island, NY). Individually indexed libraries were proportionally pooled for clustering in the cBot system (Illumina, San Diego, CA). Libraries at the final concentration of 16 pM were clustered onto a flow cell using Illumina TruSeq SR Cluster kit v3, and sequenced for 50 cycles using TruSeq SBS kit on Illumina HiSeq system. According to Illumina, for studies targeting transcription factors, ~10M reads per sample were generated.
GEO
GSM2803434