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Sample GSM2644940 Query DataSets for GSM2644940
Status Public on Jun 30, 2017
Title Estrogen_2
Sample type SRA
 
Source name metastatic breast adenocarcinoma: pleural effusion
Organism Homo sapiens
Characteristics tissue: mammary gland/breast
tumor type: breast adenocarcinoma
treatment: Estrogen
Extracted molecule nuclear RNA
Extraction protocol 5 ×10^6 nuclei were used for each run-on reaction, 2 biological replicates were produced for both vehicle and estrogen treatments. Br-UTP was incorporated into on-going transcription by run-on reaction which was performed at 30 degree for 5 min. Total RNA was extracted with TRIzol and fragmented with RNA Fragmentation Reagent. Fragmented RNA was purified with P-30 column, which was followed by T4 polynucleotide kinase treatment to dephosphorylate the 3’ end of RNA fragments. Br-UTP labeled RNA was enriched twice with anti-BrdU beads and precipitated overnight.
PolyA tailing was done using E.coli Poly(A) Polymerase, followed by reverse transcription with oNTI-223-index: /5Phos/GATCGTCGGACTGTAGAACTCTGAAC/iSp18/TCAGACGTGTGCTCTTCCGATCTTTTTTTTTTTTTTTTTTTTVN which allows custom barcoding. Exonuclease I was used to remove excess oligo after reverse transcription. DNA-RNA duplex was purified with ChIP DNA Clean & Concentrator Kit followed by RNAse H treatment. cDNA was circularized amplified with oNTI-201: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGACG and oNTI-200: CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT(XXXXXX is barcode used for specific sample) for 12 to 14 cycles. Final PCR product was purified by running 10% TBE gel and cleaned up.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2000
 
Description analyzeRNA_SX_noadj.txt
analyzeRNA_SX_rpkm.txt
Data processing Library strategy: Gro-seq
Reads were first trimmed to remove 3´polyA with Homer 4.6
Trimmed reads were quality filtered with FASTX Toolkit 0.0.14 with options -v -q 10 -p 97 to have minimum 97% of reads with quality score greater than 10
Ribosome RNA reads were removed with Bowtie 1.12. Then filted reads were aligned to hg19 using Bowtie 1.12 with options v = 2, m = 3, k = 1.
Aligned files (sam files) were made into Tagdirectories with Homer 4.6. Raw reads counts calculated with analyzeRNA.pl command, option -noadj to specify raw counts. Normalized expression files were created also with analyzeRNA.pl command, with option -rpkm to specify the normalization.
Differential gene expression analysis was performed with edgeR 3.8.5
Genome_build: hg19
Supplementary_files_format_and_content: analyzeRNA_SX_noadj.txt tab-delimited text files include raw reads values for each sample, and analyzeRNA_SX_rpkm.txt tab-delimited text files include RPKM values for each sample.
 
Submission date May 31, 2017
Last update date May 15, 2019
Contact name Shixiong Wang
E-mail(s) [email protected]
Phone +47-22845865
Organization name University of Oslo
Department Centre for Molecular Medicine Norway
Street address Gaustadalléen 21
City Oslo
ZIP/Postal code 0349
Country Norway
 
Platform ID GPL11154
Series (1)
GSE99508 Genome wide analysis of the transcription induced by estrogen and tamoxifen in MCF-7 cells
Relations
BioSample SAMN07180821
SRA SRX2873444

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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