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| Status |
Public on Aug 22, 2017 |
| Title |
IP_H3K9me2_24335_1 |
| Sample type |
SRA |
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| Source name |
24335
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: H3K9me2 (anti-H3K9me2, 5 ul per IP, Abcam, catalog no. ab1220)
cell type: NMC
cell line: ZNF532-NUT 24335
agent: none
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| Treatment protocol |
BioTAP-XL procedure and mass spectrometric data analysis:7.5 × 108 cells grown as monolayer cultures in 150mm dishes (50 plates total) were used for BioTAP-XL purification. Tetracycline was added (1 μg/mL) to 60-70% confluent culture to induce transcription of the N- or C-BioTAP tagged BRD4-NUT cDNA for 24h or for 4d in the case of ZNF532 cDNA. Recovery and analysis of nascent RNA: 4 × 106 ZNF532-NUT 24335 cells were grown in 100 mm dishes. Cells were split into two T-25 flasks and JQ1 (10uM final concentration) or DMSO were added to the media. After 4 hours, cells were harvested and nascent RNA was purified as described (3). Each RNA library was constructed from 50 ng of nascent RNA following the manufacturer’s instructions (NEBNext Ultra directional RNA library prep kit, New England BioLabs)
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| Growth protocol |
293TRex, TC-797 (31), and BICR6 (Hypopharyngeal squamous cell carcinoma (32) were maintained in Dulbecco Modified Eagle medium (DMEM) (Invitrogen) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, S10350, Atlanta Biologicals), 1% pen-strep and 1X GlutaMAX (Invitrogen). NMC cells 24335 (ZNF532-NUT) were initially established in OCMI-E media (33) (purchased from Live Tumor Culture Core, University of Miami, http://sylvester.org/shared-resources/Live-Tumor-Culture-Core). 797TRex and 293TRex derivatives, which contain a single genomic FRT recombination site, and constitutively express tetracycline repressor (TR), were created as described (2) and maintained as above with blasticidin (Invitrogen) and zeocin (Invitrogen). 797TRex, 293TRex and /N- and C- BioTAP-BRD4-NUT lines were generated as described previously (2-3), and maintained in blasticidin and hygromycin (Sigma). Stable 797TRex and BICR6 ZNF532-BioTAP lines were generated by lentiviral transduction using the following vectors: N-BioTAP and C-BioTAP pHAGE-CMV-TET-ZNF532 (details on vectors and constructs will be provided upon request) followed by selection with puromycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The main steps of BioTAP-XL procedure: harvesting cells, formaldehyde cross-linking, chromatin preparation, affinity purification, input and IP protein recovery were performed as described (12, 36). Cross-linked chromatin for ChIP-seq experiments using antibodies against proteins and histone modifications was prepared from 5 × 10(8) 24335 (ZNF532-NUT) NMC cells as described (36). Nascent RNA was purified as described (3). Each RNA library was constructed from 50 ng of nascent RNA following the manufacturer’s instructions (NEBNext Ultra directional RNA library prep kit, New England BioLabs)
ChIP-seq library preparation were performed as described (Alekseyenko et al. 2015). RNA library was constructed from 50 ng of nascent RNA following the manufacturer’s instructions (NEBNext Ultra directional RNA library prep kit, New England BioLabs)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
IP_H3K9me2_24335_1
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| Data processing |
Reads were aligned to the human reference genome (GRCh38 assembly) using Bowtie (version 0.12.5), retaining only uniquely mapped reads.
Smoothed enrichment profiles were generated using the SPP package (Kharchenko et al. 2008), using smoothing bandwidth of 5kb.
Genome_build: GRCh38
Supplementary_files_format_and_content: tdf files were generated by converting wig files using igvtools
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| Submission date |
Mar 17, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Kharchenko |
| Organization name |
Harvard Medical School
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| Department |
DBMI
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| Lab |
Kharchenko
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| Street address |
10 Shattuck St.
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE96775 |
Chromatin-associated protein interactions drive megadomain formation in NUT midline carcinoma |
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| Relations |
| BioSample |
SAMN06616212 |
| SRA |
SRX2652423 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2543134_H3K9me2_1st.tdf |
246.1 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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