title: GSM3141514: ES cells Asf1a KO repeat 2; Mus musculus; RNA-Seq
library_construction_protocol: Total mRNA were extracted using the miRNeasy Mini kit (Qiagen). Cells for ChIP-seq were cross-linked with 1% formaldehyde (final concentration) for 10 min and quenched with 125 mM glycine for 5 min at room temperature and washed once with TBS. The pellets 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 divided into two aliquots and washed with MNase digestion buffer (20 mM Tris-HCl, pH7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2). After re-suspending in 500 μL MNase digestion buffer containing a proteinase inhibitor cocktail (Sigma), the lysates were incubated in the presence of 1,000 units of MNase (NEB, Ipswich, MA, Cat.# M0247S) at 37 °C for 20 min with continuous mixing in thermal mixer (Fisher Scientific, Pittsburgh, PA). After adding the same volume 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 15 min (30 secs on / 30 secs off) using Bioruptor Twin (UCD-400) (Diagenode) 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). The chromatin was then incubated with 5 µg of rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling, Cat.# 9733), 2 µg of rabbit plolyclonal H3K4me3 antibody (Abcam, Ab8580) on a rocker overnight. Protein G beads were added for 3 hours incubation. 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), 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 PCR purification kit (Qiagen) after the treatment of RNase A and proteinase K. RNA-seq libraries and deep sequencing were conducted by Novogene mRNA next-generation sequencing services. Two replicates for each sample were sequenced. ChIP-seq libraries were prepared from 10 ng ChIP and input DNA using the Ovation ultralow DR Multiplex kit (NuGEN). The ChIP-seq libraries were sequenced at single end on an Illumina HiSeq 2000 instrument.
attributes.tag: GEO Accession
attributes.value: GSM3141514