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| Status |
Public on Jul 02, 2018 |
| Title |
NSC H3K27me3 ChIP-seq spike-in |
| Sample type |
SRA |
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| Source name |
whole cell
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| Organism |
Homo sapiens |
| Characteristics |
media: DMEM
cell cycle stage: asynchronization
chip antibody: Cell Signaling, Cat.# 9733
cell_line/tissue: NSC
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| Growth protocol |
SF7761 and SF8628 cell lines from patients harboring the histone H3.3 K27M mutation were obtained from Hashizume et al. (2012). NSCs (N7800-100) were purchased from Invitrogen and cultured and maintained in NSC medium (A10509-01, StemPro NSC SFM, Invitrogen).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen tissues were cut, divided into 50 mg aliquots, and stored in tubes at -80°C. The frozen tissues were homogenized on ice for 15–30 seconds in 500 μL 1X PBS using a tissue grinder (ACTGene). Tissue homogenates or cultured cells 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). For the normalization of ChIP efficiency, S2 or Sf9 chromatin was added. The chromatin was then incubated with 5 µg of rabbit monoclonal anti-Ezh2 antibody (Cell Signaling, Cat.# 5246), 5 µg of rabbit monoclonal anti-H3K27me3 antibody (Cell Signaling, Cat.# 9733), 2 µg of rabbit polyclonal anti-H3K27M antibody (Millipore, Cat.# abe419), or 5 µg of mouse monoclonal anti-Jarid2 antibody (Novus, Cat.# NB100-2214) on a rocker overnight. Protein G-magnetic beads (30 μl, Life Technologies) 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.
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 to 51 base pairs from both ends on an Illumina HiSeq 2000 instrument in the Mayo Clinic Center for Individualized Medicine Medical Genomics Facility.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
spike-in Drosophila reference chromatin
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| Data processing |
For the spike-in H3K27me3 ChIP-seq, the sequencing reads were aligned to a combined hg19/dm6 or h19/sf9 genome and reads were separated into each organism post-alignment. For other normal ChIP-seq, paired-end reads were aligned to the human genome (hg19) using the Bowtie2 software. For the further analysis, the consistent pair reads were used.
The genome-wide read coverage was calculated by BEDTools and in-house Perl programs.
ChIP-seq peaks were identified by MACS2 with the parameter of broad peak calling and the p value cutoff is set to 1 x 10-5.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files were generated, scores represent the normalization reads density.
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| Submission date |
Feb 13, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
zhiguo zhang |
| E-mail(s) |
zz2401@cumc.columbia.edu
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| Phone |
212-851-4936
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| Organization name |
Columbia University
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| Department |
Pediatric and Genetics and Development
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| Lab |
Irving Cancer Research Center
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| Street address |
1130 St. Nicholas Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10032 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE94834 |
H3.3K27M mutant proteins reprogram epigenome by sequestering the PRC2 complex to poised enhancers |
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| Relations |
| BioSample |
SAMN06328388 |
| SRA |
SRX2556724 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2486317_k89.spikenorm.bw |
255.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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