 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jul 14, 2015 |
| Title |
3hTET_293TB4Nf |
| Sample type |
SRA |
| |
|
| Source name |
293T
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: 293Trex (cells originating from embryonic kidney)
cell line: 293TB4Nf
treatment: 3h -incubation time period with Tetracycline (final concentration of 1ug/ml).
|
| Treatment protocol |
2 × 106 293Trex cells were grown in 100 mm dishes. Tetracycline was added to the media to a final concentration of 1ug/ml. 2 × 106 TC-797 and 1015 cells were grown in 100 mm dishes. JQ1 was added to the media to a final concentration of 500 nM for TC-797 cells, or 2 uM for 1015 cells.
|
| Growth protocol |
293TRex Flag-BRD4-NUT-HA cell lines were 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 (above) supplemented with Blasticidin (Invitrogen) and Hygromycin (Sigma). 293TRex 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). PER-403 (Kees et al. 1991) were maintained in DMEM with 20% FBS and supplemented as above. 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.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Nascent RNA purification was based on the Nascent-seq protocol (Khodor et al. 2011; Ferrari et al. 2013). Briefly, cells were harvested and homogenized 3 times with a 25G needle in 1ml buffer CSK buffer (15mM HEPES pH 7.8; 100mM NaCl; 3mM MgCl2; 1mM EGTA; 500mM Sucrose; 0.5% Triton X-100; Complete protease inhibitor (Roche) and 0.5x SUPERaseIN (Life technologies) to isolate the nuclei. Nuclei were collected by centrifugation and resuspended in 80ul of CF buffer (10mM Tris pH 8.0; 100mM NaCl; 25mM EDTA; 1x SUPERaseIN (Life technologies). Immediately afterwards, 1ml of NUN buffer (1.5M UREA; 20mM HEPES pH 7.8; 300mM NaCl; 7.5mM MgCl2; 500mM EDTA; 1% NP40) was added, and the sample was vortexed to lyse the nuclei. The mixture was incubated on ice with occasional agitation for 10 min to allow precipitation of genomic DNA and core histones together with engaged RNA Pol II and nascent transcripts. After spinning the sample at 20000g for 10 min at 4°C, the pellet was washed twice with NUN buffer. Next, the pellet was resuspended in 700 ul of CF buffer supplemented with 1% SDS and 1 mg/ml Proteinase K using a 25G needle and incubated at 55°C for 60 min. The lysate was extracted with Phenol/Chloroform. Nucleic acids were precipitated, dissolved in H2O and treated with Turbo DNaseI (Ambion) for 60 min at 37°C to eliminate DNA contamination.
50 ng of nascent RNA was used to make the RNA library for Illumina following the instructions provided by manufacture for NEBNext Ultra directional RNA library prep kit (New England BioLabs).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Nascent RNA
|
| Data processing |
Nascent RNA-Seq reads were first aligned to the human reference genome using Bowtie (version 0.12.5) with the maximum insert size (-X) set to 100,000, keeping only uniquely mapped reads.
HTSeq (Anders et al. 2015) was used to count reads mapped to genes (using GENCODE v21 annotation).
DESeq (Anders and Huber 2010) was subsequently employed to estimate nascent transcription levels, and analyze differential expression between TC-797 following and prior to JQ1 treatment, and between 293TRex following and before BRD4-NUT induction.
tdf files were generated by converting wig files using igvtools
Genome_build: hg38
Supplementary_files_format_and_content: TDF files (for IGV browser) containing smoothed read density
Supplementary_files_format_and_content: RNASeq.counts.txt reports the read count estimates per gene for each sample
|
| |
|
| Submission date |
Jul 14, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Kharchenko |
| Organization name |
Harvard Medical School
|
| Department |
DBMI
|
| Lab |
Kharchenko
|
| Street address |
10 Shattuck St.
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (2) |
| GSE70868 |
The oncogenic BRD4-NUT chromatin regulator drives aberrant transcription within large topological domains |
| GSE70871 |
Recovery and analysis of nascent RNA |
|
| Relations |
| BioSample |
SAMN03857576 |
| SRA |
SRX1094533 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1820986_3_RNA_3hTET_293TB4Nf_TTAGGC_L004.minus.tdf |
181.7 Mb |
(ftp)(http) |
TDF |
| GSM1820986_3_RNA_3hTET_293TB4Nf_TTAGGC_L004.plus.tdf |
181.3 Mb |
(ftp)(http) |
TDF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
