GSM8670941: HL60, wildtype, untreated, EGR1 (ChIP); Homo sapiens; ChIP-Seq
ChIP-seq with antibodies for EGR1 (Bethyl), H3K27ac (Abcam), and H3K4me1 (Abcam) were done in HL-60 cells.  For each experiment, 10 million cells were collected and crosslinked in growth medium containing 1% formaldehyde for 5 minutes then quenched in 0.125M Glycine. Cells were then washed twice with PBS then flash frozen. Cell pellets were lysed in ChIP buffer (150mM NaCl, 1% TritonX-100, 5mM EDTA, 10mM TrisCl, 0.5mM dithiothreitol (DTT), and 0.7% SDS) plus protease inhibitors on ice for 10 minutes.  Sonication was performed using the Covaris ME220 Focused-ultrasonicator to reach an average chromatin fragment length of 200-400bp. Sheared chromatin was cleared by centrifugation at 13,000 RPM for 10 minutes. Prior to adding antibody, 5% of the sample was collected as an input control. For 10 million cells, 10ug of antibody (3ug for H3K27ac) and protein A or G Dynabeads (Invitrogen) were added to chromatin. For RNAPII ChIP-seq, a custom rabbit polyclonal antibody raised against the N-terminal domain was used. On Day 2, beads were washed with twice with Mixed Micelle Wash Buffer (150mM NaCl, 20mM Tris-HCl, 5mM EDTA, 5.2% sucrose, 1% Triton X-100, 0.2% SDS), Buffer 500 (500mM NaCl, 25 mM HEPES, 10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate), LiCl/detergent Wash (250 mM LiCl, 10 mM Tris-HCl, 1 mM EDTA, 0.5% Na deoxycholate, 0.5% NP-40) and once with 1× Tris-EDTA (TE) buffer. Samples were then incubated in TE plus 1% SDS at 65°C for 10 minutes, rotating at 1200 RPM. Elution was repeated twice, and the eluates were further incubated overnight at 65°C to reverse cross-linking, along with the untreated input. On Day 3, proteinase K (0.5mg/mL final concentration) was added to the samples and they were incubated for 1 hour at 65°C. The Zymo ChIP DNA Clear Concentrator Kit (Zymo research) was used for the final DNA extraction. Sequencing libraries were prepared with the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB).