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Sample GSM3127596

Query DataSets for GSM3127596

Status

Public on May 03, 2018

Title

T3 FFPE mRNA-seq (500ng)

Sample type

SRA

Source name

breast cancer tumor sample

Organism

Homo sapiens

Characteristics

tissue: breast cancer tumor sample
sample_type: FFPE
protocol: mRNA-seq
starting material (ng): 500
patient: T3
archival time: 4-5 years

Extracted molecule

total RNA

Extraction protocol

FFPE tumor samples were sectioned to 5 micron slices and deparafinized at 90°C for 5 min. Total RNA was extracted using nucleic acid isolation kit (AllPrep® Qiagen) according to the protocol instructions, with the following modifications: DNAse treatment was at 37° for 20 minutes and the column was incubated for 1 min before each washing step with buffer RPE. Samples were eluted in 30 μL purified water. RNA from FF tumor samples was extracted using Trizol. RNA concentrations were determined by Qubit™ fluorometer (Thermofisher scientific) and the RNA quality was determined by measuring the RNA integrity number (RIN) using Agilent TapeStation.
Total RNA from FFPE and FF samples was processed using two protocols: 1. Truseq RNA Sample preparation kit v2 (illumina) (cat# RS-122-2002) for mRNA-seq libraries. Briefly, polyA fraction (mRNA) was purified from 500ng or 100ng of total RNA following by fragmentation and generation of double stranded cDNA. Then, end repair, A base addition, adapter ligation and PCR amplification steps were performed. 2. Truseq Stranded total RNA with Ribo-Zero Gold. Library Protocol: TruSeq® Stranded Total RNA (Illumina) (Cat # RS-122-2301, RS-122-2302) for ribosomal-depletion libraries. Briefly, After rRNA depletion from 500ng total RNA with rRNA Ribo-Zero Gold removal mix, cDNA was performed followed by second strand synthesis with dUTP instead of dTTP. Then, A base addition, adapter ligation, UDG treatment and PCR amplification steps were performed

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Fragments were mapped to the human genome (hg19) using TopHat version V2.0.5
Only uniquely mapped fragments to the genome were considered and exonic and intronic signals were calculated using HTSeq
The signals of the same sample from all lanes were summed
The number of fragments obtained for each gene in each sample was normalized to the total number of fragments obtained from this sample
The minimum expression level threshold was set to 5 (log2 scale) to reduce noise (based on data distribution).
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include normalized number of reads (log2 scale) for each sample

Submission date

May 02, 2018

Last update date

May 04, 2018

Contact name

Noa Bossel Ben-Moshe

Organization name

Weizmann

Street address

Hertzl Street

City

Rehovot