GSM9723847: C2,Mithra,rep1,EGR1; Homo sapiens; ChIP-Seq
ChIP-seq was performed as per the manufacturer's protocol (CST, catalog #: 9003) with modifications. Briefly, Lf-C2 and Lf-4-2 cells were treated with either DMSO or 300 nM Mithramycin A for 24 hours. Cells were crosslinked with Cross-link Gold (Diagenode, catalog #: C01019027), followed by 1% formaldehyde for 10 mins at room temperature. Fixed cells were enzymatically digested and sonicated. 750 µg of protein was incubated with anti-EGR1 (Bethyl, catalog #: A303-390A, 45 µg) or control IgG (CST, catalog #: 2729) overnight at 4°C with rotation. The antibody-bound chromatin was immunoprecipitated by protein G magnetic beads and eluted. After reversal of the crosslink, the DNA was purified, and sequencing was performed at the OncoGenomics Shared Resources (OGSR) of the Sylvester Comprehensive Cancer Center. ChIP-seq libraries were prepared using the KAPA HyperPrep Kit (Roche, Cat # 07962363001). In brief, immunoprecipitated DNA fragments (IP frags) were quantified and qualitatively assessed on the ThermoFisher QuBit Fluorometer's High Sensitivity DNA (cat. Q32855) and Agilent Femto Pulse Ultra Sensitivity NGS kits (cat. FP-1101-0275), respectively. Input of IP frags. were between 0.17 and 25 ng. HyperPrep kit was used for end repair, A-tailing and adapter ligation followed by PCR amplification. Finished libraries were quantified and qualitatively assessed on the ThermoFisher QuBit using Fluorometer's High Sensitivity DNA (Cat #Q32855) and Agilent Fragment Analyzer High Sensitivity NGS kits (Cat # DNF-474-0500) prior to normalization and pooling in preparation for sequencing.. Pooled libraries were sequenced using paired-end 150 base pair chemistry on an Illumina NovaSeq X Plus instrument. Targeted average sequencing depth for ChIP-seq broad peak was 30 million clusters per library.