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| Status |
Public on May 21, 2018 |
| Title |
GRO-seq_231ERαWT_0min_rep2 |
| Sample type |
SRA |
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| Source name |
GRO-seq_231ERαWT_0min
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| Organism |
Homo sapiens |
| Characteristics |
cell line background: MDA-MB-231
transduced with: ERα wt
treated with: E2, 100 nM, 0 min
molecule subtype: nascent RNA
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| Treatment protocol |
Cells were treated with vehicle (EtOH) for 45 min or 100 nM E2 for 20 or 45 min.
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| Growth protocol |
MDA-MB-231 cells obtained from ATCC were transduced with ERα wile-type or L540Q mutant by lentivirus-mediated transduction and maintained in phenol red-free Dulbecco’s modified Eagle’s medium/nutrient mixture F-12 Ham (DMEM/F-12; Sigma, D2906) supplemented with 10% charcoal-dextran treated calf serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 2 x 106 cells were seeded on a 15 cm2 plate and grown to ~80% confluence. The cells were washed twice with ice-cold PBS and collected by scraping in Swelling Buffer [10 mM Tris•HCl pH 8.0, 2 mM MgOAc, 3 mM CaCl2, 0.5 mM DTT, 1x complete protease inhibitor cocktail, and SUPERase•In (Ambion)] on ice. The cells were pelleted by centrifugation, resuspended in Lysis Buffer [10 mM Tris•HCl pH 8.0, 2 mM MgOAc, 3 mM CaCl2, 10 mM NaCl, 0.5% NP-40, 300 mM sucrose, 0.5 mM DTT, 1x complete protease inhibitor cocktail, and SUPERase•In (Ambion)]. To isolate the nuclei, the swollen cells were lysed by pipetting up and down 60 times using P1000 pipet tips. The nuclei were pelleted by brief centrifugation, equilibrated and dispersed in Freezing Buffer to 5 x 106 nuclei/100 μL [50 mM Tris•HCl pH 8.3, 5 mM MgCl2, 40% glycerol, 0.1 mM EDTA, and SUPERase•In (Ambion)], aliquoted, and stored at -80°C.
The nuclei were incubated in the presence of 5’-bromo-UTP and α-32P radiolabeled-CTP for 5 min., with subsequent quenching by the addition of RQ-1 DNase (Promega, M6101). RNA was isolated from the run-on reaction mixture and hydrolyzed by incubating with 0.2 N NaOH on ice for 15 min. The fragmented RNA was treated with T4 PNK (NEB, M0201S) in the absence of ATP to dephosphorylate the 3’-terminus. Nascent transcripts were isolated from the total RNA with two rounds of affinity purification using anti-BrdU antibody-conjugated agarose beads (Santa Cruz Biotechnology, sc-32323). PolyA tails were added to the nascent transcripts using 1 mM ATP and 0.5 U/μL E. coli Poly(A) Polymerase (NEB, M0276S) at 37°C for 8 min. cDNA was generated using the oNTI223HIseq primer (custom synthesized by IDT, HPLC purified) (Ingolia, 2009) and purified on an 8% polyacrylamide TBE-urea gel. The purified cDNA was then circularized using CircLigase (Epicentre, CL4111K), relinerized using APE1 (NEB, M0282S), and amplified using TruSeq small RNA-seq PCR primers (custom synthesized by IDT, HPLC purified) to generate the GRO-seq library. The library quality was assessed using a D1000 ScreenTape (Agilent, 5067-5582) on a 2200 TapeStation (Agilent) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851). The libraries with unique adaptor barcodes were multiplexed and sequenced on an Illumina HiSeq 2000 (single-end, 50 bp reads) for ~18 million raw reads per biological replicate.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
library strategy: GRO-seq
FastQC
Adaptor trimming using Cutadapt
Alignment to the human reference genome (hg19) and one complete copy of an rDNA repeat (GenBank ID: U13369.1) using the BWA aligner. The replicates 1, 2 are biological replicates and replicates 3, 4 are technical replicates of replicates 1,2 respectively. The technical replicates (1,3 & 2,4) were merged to consider as a single replicate after confirming positive correlation between them and used for further analysis.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files for visualization
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| Submission date |
Feb 21, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
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| Organization name |
UT Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE95122 |
Priming and Activation of ERα Enhancers Through Ordered and Cooperative Interactions Between p300 and Mediator [GRO-seq] |
| GSE95123 |
Priming and Activation of ERα Enhancers Through Ordered and Cooperative Interactions Between p300 and Mediator |
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| Relations |
| BioSample |
SAMN06350773 |
| SRA |
SRX2578923 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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