GSM2945855
GSM2945855: WT IMM_H3K9ac; Mus musculus; ChIP-Seq
Thymi were harvested, processed for single cell suspension, and stained with antibody for cell sorting by FACS Aria.  Sorted samples were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature. After washing with TBS, cells were resuspended in cell lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) and incubated on ice for 10 min. Lysates were washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After resuspendsion in 200 μL fresh MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated with 2,000 units of MNase (NEB, Cat # M0247S) per 4 x 106 cells at 37 °C for 20 min with continuous mixing in thermal mixer. After adding 200 μL of sonication buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), the lysates were sonicated for 30 min (30 sec on / 30 sec off) using Bioruptor Twin (UCD-400) (Diagenode, Inc.) and centrifuged at 21,130 x g for 10 min. The supernants were collected, and the chromatin content was estimated by the Qubit assay (Invitrogen). For normalization of ChIP efficiency, Drosophila chromatin equivalent to about 5% of total chromatin was added. The chromatin was then incubated with anti-H3K27ac antibody (Abcam, Cat.# ab4729, lot# GR150367) or in-house generated anti-H3K9ac antibody (EDL lot 1) on a rocker overnight. Protein G-magnetic beads (30 μL, Life Technologies) were added, and further incubated for 3 hours. The beads were extensively 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), LiCl2 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 PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. ChIP-seq libraries were prepared from about 5 ng ChIP and input DNA using the ThruPLEX® DNA-seq Kit V2 (Rubicon Genomics, Ann Arbor, MI) according to manufacturere protocol.
GEO
GSM2945855
GEO Accession
GSM2945855