GSM4971610: RH4 - PAX3-FOXO1 ChIP - rep1; Homo sapiens; ChIP-Seq
For spike-in normalized ChIP-seq, human RMS and Dbt/iP3F 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 FOXO1 (Cell Signaling #2880), H3K27ac (Active Motif #39133), H3K27me3 (Active Motif #39155), BRD9 (Abcam ab137245), or DPF2 (Abcam ab134942). Input and ChIP-enriched DNA was decrosslinked and purified using Qiagen MinElute PCR Purification columns prior to library preparation. For ATAC-seq, 5x104 cells per experiment were first washed with RSB buffer and gently permeabilized with RSB lysis buffer on ice. Cells were suspended in 50 uL of tagmentation master mix prepared from Illumina Tagment DNA TDE1 Enzyme and Buffer Kit components (#20034197), and transposition was performed for 30 minutes at 37°C. Tagmented DNA fragments were isolated using Qiagen MinElute PCR Purification columns prior to library preparation. 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 for 14 cycles and isolated from unreacted primers by gel purification. ATAC-seq libraries were amplified with barcoded Nextera primers for 14 cycles and excess primers were removed by size selection with AMPure XP beads. Libraries were sequenced on the HiSeq4000 platform running in PEx150bp mode.