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| Status |
Public on Jul 14, 2015 |
| Title |
IP_H3K36me3_797_Rep1_Jun2014 |
| Sample type |
SRA |
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| Source name |
797
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: H3K36me3 (anti-H3K36me3, 3 ul per IP, Abcam , catalog no. ab9050)
cell type: NMC
cell line: TC-797 (Toretsky et al. 2003)
treatment: none
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| Treatment protocol |
293TRex cells, which contain a single genomic FRT recombination site, were maintained with Blasticidin (Invitrogen). Flag-BRD4-NUT-HA cell line was generated by recombination with the plasmid Flag-Brd4-NUT-HA using the Flp-In technology manufacturer’s instructions (Invitrogen). The resulting cell lines were maintained in medium supplemented with Blasticidin (Invitrogen) and Hygromycin (Sigma). pEGFPC1-BRD4-NUT was created as described (French et al. 2008) and transfected into 293T cells using Lipofectamine 2000 according to the manufacturer's instructions (Life Technologies). Tetracycline was added (1 μg/mL) to 60-70 % confluent culture to induce transcription Flag-BRD4NUT for 2h, 3h and 7h. JQ1 was added to the media to a final concentration of 500 nM for TC-797 cells
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| Growth protocol |
293Trex cells and NMC cell lines, TC-797 (Toretsky et al. 2003), 1015 (Grayson et al. 2014), 10326 (French et al. 2008) 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). PER-403 (Kees et al. 1991) were maintained in DMEM with 20% FBS and supplemented as above. 293TRex-Flag-BRD4NUT cells 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).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Formaldehyde cross-linked chromatin for ChIP-seq experiments with antibodies against proteins and histone modifications were prepared from 1.5 × 108 cells of each NMC cell line and 293TRex-Flag-BRD4NUT-HA as described (Alekseyenko et al. 2015). Chromatin from 0.8g of 1015 patient tissue ((Grayson et al. 2014) was prepared as follows: frozen tissue was ground to a powder with a ceramic mortar and pestle chilled with liquid N2. The powder was immediately transferred into a 50 ml Falcon tube filled with 9mL of NEB buffer (10% sucrose, 20 mM HEPES at pH 7.6, 10 mM NaCl, 3 mM MgCl2, 0.5% Triton, 0.1 mM PMSF). The mixture was vortexed for 15sec to brake-up the clumps, and immediately after, 45 mL of PBS and 0.5mL of 37% formaldehyde were added to the tube. The resulting mixture was incubated for 25 min at 25°C on an orbital shaker platform with gentle mixing (150 rpm). Washing and chromatin shearing steps were performed as described (Alekseyenko et al. 2015). Affinity purification was performed as described (Kharchenko et al. 2011). Input and IP DNA recovery and ChIP-seq library preparation were performed as described (Alekseyenko et al. 2015). The following antibodies against proteins and histone modifications were used for ChIP-seq experiments: anti-H3K27ac (10 ul per IP, Active Motif, catalog no. 39133); anti-H3K9ac (15 ul per IP, Abcam, catalog no. ab12179); anti-H3K14ac (2 ul per IP, Abcam , catalog no. ab52946), anti-H3K36me3 (3 ul per IP, Abcam , catalog no. ab9050) anti-P300 (10 ul, Bethyl, catalog no. A300-358A); anti-BRD4-SL (against short and long isoforms of BRD4, 5 ul per IP, Bethyl, catalog no. A301-985A), anti-BRD4-L (against long isoforms of BRD4, 15 ul per IP, Active Motif, catalog no. 39909); anti-NUT (15 ul per IP; Cell Signaling, catalog no. 3625).
ChIP-seq library preparation were performed as described (Alekseyenko et al. 2015).
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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_H3K36me3_797_Rep1_Jun2014
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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.
tdf files were generated by converting wig files using igvtools
Genome_build: GRCh38
Supplementary_files_format_and_content: TDF files (for IGV browser) containing conservative estimates of smoothed enrichment
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| Submission date |
Jul 14, 2015 |
| 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 (2) |
| GSE70868 |
The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains |
| GSE70870 |
Chip-seq mapping of active chromatin marks in BRD4-NUT and different NMC cells |
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| Relations |
| BioSample |
SAMN03857521 |
| SRA |
SRX1094487 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1820940_IP_H3K36me3_797_Rep1_Jun2014_VS_Input_797_Rep1_Jun2014.smoothed.enrich.tdf |
260.3 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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