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Sample GSM3121612

Query DataSets for GSM3121612

Status

Public on Oct 01, 2018

Title

SH_TS_BC59

Sample type

SRA

Source name

Breast cancer

Organism

Homo sapiens

Characteristics

gender: female
dmfs time: NA
dmfs event: NA
sample type: Solid tumor
irdm subtype: LumA
pam50 subtype: LumA
immunity group: iweak
proliferation group: low
er status rna: ER+
pr status rna: PR+

Extracted molecule

total RNA

Extraction protocol

FFPE tissues total RNA extraction was routinely performed using Roche FFPE RNA extraction kit according to the manufacturer’s protocol.
The Illumina TruSeq Targeted RNA expression kit was used to build libraries of the targeted 72-genes. To synthesize cDNA, 200 to 800 nanograms of purified FFPE RNA in a total volume of 3 µl was mixed with 4.0 µl RCS1, 2.0 µl ProtoScrip II Reverse Transcriptase, 1.0 µl 10mM DTT at 42OC for 30 min and 94OC for 10 min. The cDNA was hybridized with custom oligo pools in a thermal cycler programed to gradually decrease temperature from 70 OC to 30 OC in 30 minutes. The RNA/Oligo hybrid products were washed, extended and ligated. The ligated DNA was amplified by DNA polymerase on the thermal cycler with 35 PCR cycles of 98 OC for 30 seconds, 62 OC for 30 seconds and 72 OC for 60 seconds. The PCR products were purified with AMPure XP beads and eluted in 15 µl of buffer, measured using Agilent Bioanalyzer2100 and DNA1000 chips, pooled with equal amounts of DNA from each sample’s library, and finally diluted to 4 nM, denatured, and loaded to Nextseq500 according to the manufacturer’s protocol.
Libraries were multiplexed and sequenced on Illumina Nextseq500 to generate 75bp single read R1.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

FFPE RNA
Targeted RNA-seq
SH_TS_BC59

Data processing

Illumina Casava1.7 software used for basecalling and sequencing data were demultiplexed based on dual indexes.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, demultiplexed and then only read 1 in each sample were mapped to known Targeted regions of human genomes for counts using R package "ShortRead".
Raw counts of all samples were imported to edgeR for normalization by the size of the transcripts and by the size of the library and then calculation of CPM per sample as a gene expression matrix
Gene expression CPM dataset were median-centered and column-standardized using DWD
DWD dataset were used for iRDM data analysis including subtype classification, iRDM risks, immunity scores, and proliferation scores.
Genome_build: hg19
Supplementary_files_format_and_content: csv files were generated including raw counts and normalized gene expression data

Submission date

Apr 30, 2018

Last update date

Oct 01, 2018

Contact name

Zhiyuan Hu

E-mail(s)

HUZI1111@OUTLOOK.COM

Phone

9195274295

Organization name

University of North Carolina at Chapel Hill