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| Status |
Public on Aug 15, 2013 |
| Title |
siCTL_minusDHT_hs26l2_1 |
| Sample type |
SRA |
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| Source name |
LNCaP cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: LNCaP cells
transfected with: siCTL
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| Treatment protocol |
Prior to androgen treatment, we grow the cells in UltraCULTURE (Phenol red free) + 5% Charcoal Dextran Stripped (CDS) serum for 48 hours. For androgen induction, we treat cells with DHT from a 100 uM stock in 70% ethanol to a final concentration of 100 nM
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| Growth protocol |
We maintain LNCaP cells in RPMI 1640 + 10% FBS medium. We typically grow LNCaP cells in 150 or 100 mm polystyrene tissue culture dishes depending on number of cells required. Media is changed at minimum twice per week, or before media becomes orange. Cells are split soon after they reach 80% confluence.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Nuclei were isolated from LNCaP cells with appropriate transfection and -/+ DHT treatment. GRO-seq libraries were prepared from 5 million cells based on previously published protocols (Wang et al., 2011). Libraries were sequenced on Illumina Hi-Seq 2000.
After harvesting the cells, RNA polymerases were allowed to run-on for 5 minutes at 30C (~100bp) in the presence of sarkosyl and BrUTP. The RNA was then base hydrolyzed and purified. BrUTP-labeled run-on RNA was immunopurified with anti-BrdUTP-coated agarose beads. Run-on RNA was then polyadenylated to generate primer sites for first strand cDNA synthesis. Complementary poly-T primers contain two adaptor sequences separated by an Ape1 restriction site, which after circularization and Ape1 digestion, will terminate both ends, thus permitting cDNA generation, PCR-amplification, and library generation for deep sequencing.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
GRO-seq
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| Data processing |
The image analysis and base calling were performed by using Illumina's Genome Analysis pipeline.
The sequencing reads were aligned to hg18 using Bowtie2, and we kept only 1 aligned read/ genome position for downstream analyses.
The bedgraph files were generated by using HOMER.
The sequencing reads were counted by using BEDTools.
The differential gene expression was assessed by edgeR.
Genome_build: hg18
Supplementary_files_format_and_content: BED aligned files after aligning the reads to hg18 by using Bowtie2, keeping only 1 mapped read / genome position
Supplementary_files_format_and_content: BedGraph files display of continuous-valued data in track format, and were generated by using the HOMER suite ("makeUCSCfile" command).
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| Submission date |
Jun 10, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
MICHAEL G ROSENFELD |
| E-mail(s) |
mrosenfeld@ucsd.edu
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| Organization name |
HHMI/UCSD
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE47805 |
LncRNA-dependent mechanisms of androgen receptor-regulated gene activation programs [GRO-seq I] |
| GSE47807 |
LncRNA-dependent mechanisms of androgen receptor-regulated gene activation programs |
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| Relations |
| BioSample |
SAMN02194347 |
| SRA |
SRX298680 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1159895_minusDHT_siCTL_hs26l2_1.sam.bed.gz |
121.1 Mb |
(ftp)(http) |
BED |
| GSM1159895_minusDHT_siCTL_hs26l2_1.strand+.ucsc.bedGraph.gz |
39.3 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1159895_minusDHT_siCTL_hs26l2_1.strand-.ucsc.bedGraph.gz |
36.4 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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