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| Status |
Public on Aug 04, 2022 |
| Title |
PRO-seq_siTop1_Veh_1h_exp2 |
| Sample type |
SRA |
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| Source name |
Human breast cancers
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
agent: vehicle
sirna: siTop1
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| Treatment protocol |
Briefly, ∼2 millions of MCF7 cells treated with E2 for 1 hr were washed 3 times with cold PBS and then sequentially swelled in swelling buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2) for 10 min on ice, harvested, and lysed in lysis buffer (swelling buffer plus 0.5% NP-40, 20 units of SUPERase-In, and 10% glycerol).
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| Growth protocol |
MCF7 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-β-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The resultant nuclei were washed two more times with 10ml lysis buffer and finally resuspended in 100 μl of freezing buffer (50mM Tris-HCl pH8.3, 40% glycerol, 5mM MgCl2, 0.1mM EDTA).
For the run-on assay, resuspended nuclei were mixed with an equal volume of reaction buffer (10mM Tris-HCl pH 8.0, 5mM MgCl2, 1mM DTT, 300mM KCl, 20 units of SUPERase-In, 1% sarkosyl, 250 μM A/GTP, 50 µM biotin-11-C/UTP (Perkin-Elmer) and incubated for 5 min at 30°C. The resultant nuclear-run-on RNA (NRO-RNA) was then extracted with TRIzol® LS reagent (Life Technologies, Cat# 10296-028) following manufacturer’s instructions. NRO-RNA was fragmented to ∼200-500nt by alkaline base hydrolysis on ice for 30 min and and neutralized by adding 1× volume of 1 M Tris-HCl pH 6.8, Excessive salt and residual NTPs were removed by using P-30 column (Bio-Rad, Cat# 732-6250), followed by treatment with DNase I (Promega Cat# M6101) and antarctic phosphatase (NEB Cat# M0289L). Fragmented nascent RNA was bound to 10 µl of MyOne Streptavidin C1 dynabeads (Invitrogen, Cat# 65001) following the manufacturer’s instructions. The beads were washed twice in high salt (2 M NaCl, 50 mM Tris-HCl pH 7.5, 0.5% Triton X-100, 0.5 mM EDTA), once in medium salt (1M NaCl, 5 mM Tris-HCl pH 7.5, 0.1% Triton X-100, 0.5 mM EDTA), and once in low salt (5 mM Tris-HCl pH 7.5, 0.1% Triton X-100).
Bound RNA was extracted from the bead using Trizol (Invitrogen, Cat# 15596-018) in two consecutive extractions, and the RNA fractions were pooled, followed by ethanol precipitation and PRO-seq libraries were prepared with NEBNext® Small RNA Library Prep kit (NEB, Cat# E7330).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
siTop1_Veh_1h_exp2
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| Data processing |
Library strategy: PRO-seq
Basecalls performed using CASAVA version 1.4
The sequencing reads were aligned to hg38 using Bowtie2 using very sensitive parameters.
The common artifacts derived from clonal amplification were circumvented by considering maximal three tags from each unique genomic position as determined from the mapping data.
To determine E2-dependent changes in gene body, the sequencing reads for RefSeq genes were counted over the first 13 kb of the entire gene body, excluding the 500 bp promoter-proximal region on the sense strand with respect to the gene orientation by using HOMER. EdgeR21 was used to compute the significance of the differential gene expression (FC ≥ 1.5, FDR ≤ 0.01).
PRO-seqs were normalized to 10 million tags, and HOMER was used to quantify eRNA expression by tabulating normalized tag numbers surrounding ± 1,000 bp from the center of the peaks. eRNAs with a >1.5-fold change in GRO-seq or PRO-seq signals were considered to be differentially expressed.
Supplementary_files_format_and_content: bigWig
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| Submission date |
Aug 07, 2019 |
| Last update date |
Aug 04, 2022 |
| Contact name |
Yuliang Tan |
| E-mail(s) |
yut020@ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
School of Medicine
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| Street address |
Room 345, CMM West, 9500 Gilman Drive, Mail Code 0648
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037-0648 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE135540 |
PRO-seq assay in human breast cancer MCF7 cells |
| GSE135808 |
Topoisomerase 1 Nicking-dependent HP1gamma Engagement Mediates Acute Activation of Phase-separated Enhancers |
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| Relations |
| BioSample |
SAMN12529347 |
| SRA |
SRX6673083 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4015604_siTop1_Veh_exp2.ucsc.bigWig |
99.6 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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