GSM6626416: MYOD, RH4, rep2; Homo sapiens; ChIP-Seq
For spike-in normalized ChIP-seq, human RMS cells and mouse C2C12 cells were fixed in 1% formaldehyde for 10 minutes before quenching with 125 mM glycine. Cells were combined in a ratio of 3 human:1 mouse cell equivalents for each experiment prior to sonication. Sonicated chromatin was immunoprecipitated with antibodies against H3K27ac (Active Motif #39133), H3K9me3 (Active Motif #39062), MYOD1 (CST #13812), or CTCF (Millipore #07-729). Input and ChIP-enriched DNA was decrosslinked and purified using Qiagen MinElute PCR Purification columns prior to library preparation. H3K27ac, MYOD1, and CTCF ChIP and Input DNA libraries were prepared by blunt end repair using the Lucigen End-It DNA End-Repair Kit, 3' A-tailing by Klenow fragment (3'-5' exo-) (NEB), adaptor ligation by T4 DNA ligase(NEB), and size selection on an E-Gel 2% EX agarose gel. Libraries were amplified with barcoded primers and isolated from unreacted primers by gel purification. Decrosslinked H3K9me3 ChIP and Input DNA was sonicated to enrich fragments 200-500-bp. Library prep was then performed without further size selection. Excess adapter and unused primers were removed with AmpureXP beads.  Libraries were sequenced on the HiSeq4000 or NovaSeq6000 platform running in PEx150bp mode.