GSM3177416: GATA1 WT H3K4me1_ChIPSeq_Rep1; Homo sapiens; ChIP-Seq
The total RNA was extract using QIAGEN RNeasy Mini Kit. 5x106 active growing cultured cells were harvest, suspended in 10ml PBS, and cross-linked with 1% formaldehyde (final concentration) for 10 min, and quenched with 125 mM glycine for 5 min at room temperature, and washed twice with PBS. The pellets were resuspended in 100 μl cell lysis buffer (1% Triton-X, 0.1% Sodium-Deoxycholate, proteinase inhibitor, TE) and incubated on ice for 10 min. Lysates were diluted with 900 ul TE, and sonicated for 15 min (30 sec on / 30 sec off) using Bioruptor pico (Diagenode, Inc., Denville, NJ), and centrifuged at top speed for 10 min. The supernants were collected and the chromatin content was estimated by the Qubit assay (Invitrogen). The chromatin was then incubated with 2.5 μg of rabbit polyclonal anti-H3K27ac antibody (Abcam, D5E4, 1:5000) on a rocker overnight. Protein G-magnetic beads (30 μL, Invitrogen) were added for 4 hours incubation. The bedads were entensively washed with ChIP buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 100 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), high salt buffer (50 mM Tris-HCl, pH8.1, 10 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate), LiCl buffer (10 mM Tris-HCl, pH8.0, 0.25 M LiCl2, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and TE buffer. Bound chromatin was eluted and reverse-crosslinked at 65 °C overnight. DNAs were purified using Min-Elute purification kit (QIAGEN, Valencia, CA) after the treatment of RNase A and proteinase K. RNA library preparation and sequence were done through Novogene. ChIP-seq libraries were prepared from ChIP DNA using the NEBNext® Ultra™ II DNA Library Prep Kit (NEB). The ChIP-seq libraries were sequenced to 50 base pairs on an Illumina HiSeq 4000 using pair-end mode in the Mayo Clinic Center for Individualized Medicine Medical Genome Facility.
GEO Accession
GSM3177416