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| Sample GSM2155922 | Query DataSets for GSM2155922 |
| | Status | Public on Mar 12, 2018 | | Title | T365 | | Sample type | SRA | | Source name | Primary breast tumor | | Organism | Homo sapiens | | Characteristics | tissue: breast tumorer ihc routinestain clinical reading: 3er ihc routinestain reading 2: 3er ihc routinestain reading 3: 3er ihc restain reading 1: 3er ihc restain reading 2: 3er ihc restain reading 3: 3er consensus: 3pgr ihc routinestain clinical reading: 3pgr routinestain reading 2: 3pgr routinestain reading 3: 2pgr restain reading 1: 2pgr restain reading 2: 2pgr restain reading 3: 2pgr consensus: 3her2 ihc clinreading: 3her2 ihc reading 2: 3her2 ihc reading 3: 2her2 clinical status: 1her2 sish reading 1: 1her2 sish reading 2: 1her2 sish reading 3: 1her2 consensus: 1ki67 reading 1: 5ki67 reading 2: 3ki67 reading 3: 5ki67 consensus: 5nhg reading 1 tubular: 3nhg reading 1 pleomorph: 2nhg reading 1 mitotic: 2nhg reading 2 tubular: 3nhg reading 2 pleomorph: 2nhg reading 2 mitotic: 2nhg reading 3 tubular: 3nhg reading 3 pleomorph: 2nhg reading 3 mitotic: 2nhg consensus: 2pam50 subtype: Her2 | | Extracted molecule | polyA RNA | | Extraction protocol | QIAGEN AllPrepPoly(A) mRNA is isolated from the total RNA in up to 96-well microtiter plate format by two rounds of purification with Dynabeads Oligo (dT)25 (Invitrogen) using a KingFisher Flex magnetic particle processor (ThermoScientific). Zinc-mediated fragmentation (Ambion) is performed and the fragmented mRNA retrieved using column purification (Zymo-Spin I-96 plates; Zymo). The sequencing library generation protocol is a modification of the dUTP method, which importantly retains the directionality (stranded-ness) of the sequenced RNA molecules. First strand cDNA synthesis is performed using random hexamers and standard dNTP mix followed by cleanup using Sephadex gel filtration (Illustra AutoScreen-96A plates; GE Healthcare), and second strand cDNA synthesis is performed using dUTP in place of dTTP in the dNTP-mix and cleanup using Zymo-Spin I-96 plates. The cDNA is end-repaired and A-tailed, and diluted TruSeq adapters with barcodes are ligated using a modified protocol (Illumina). Adapter-ligated cDNA is then size-selected to remove short oligonucleotides using carboxylic acid (CA) paramagnetic beads (Invitrogen) and polyethylene glycol (PEG), similar to the previously described methods, and automated on the KingFisher Flex. The second cDNA strand is digested using uracil-DNA glycosylase and the product is enriched by 12 PCR cycles (Illumina). The PCR product undergoes two cycles of size selection using CA-beads and varying concentrations of PEG, first to exclude DNA fragments >700 bp and then to exclude fragments <200 bp. Quality control is performed on control libraries using Qubit fluorometric measurement (Life Technologies) and Caliper LabChip XT microcapillary gel electrophoresis. Typically, 10-24 barcoded libraries are included in a pool and each pool is sequenced in at least one lane across dual flowcells. Paired-end sequencing of 50 bp read-length is performed on an Illumina HiSeq 2000 instrument. | | Library strategy | RNA-Seq | | Library source | transcriptomic | | Library selection | cDNA | | Instrument model | Illumina HiSeq 2000 | | Description | For pathology key, see pathology_consensus_key.xlsx | | Data processing | Base-calling using manufacturer's on-instrument software.Demultiplexing using the Picard ExtractIlluminaBarcodes algorithm.Filtering to remove reads that align (using Bowtie 2 with default parameters except -k 1 --phred33 --local) to ribosomal RNA/DNA (GenBank loci NR_023363.1, NR_003285.2, NR_003286.2, NR_003287.2, X12811.1, U13369.1), phiX174 Illumina control (NC_001422.1), and sequences contained in the UCSC hg19 RepeatMasker track (downloaded March 14, 2011).Remaining reads are aligned using TopHat2 2.0.5 (default parameters except for --mate-inner-dist (average size with adapters 355, range 268-465, measured for each sample individually) --mate-std-dev 100 --library-type fr-firststrand --keep-fasta-order --no-coverage-search) to the human genome reference GRCh37/hg19 (with b37 masked chromosome Y and hs37d5 decoy sequences) together with 80,883 transcript annotations from the UCSC knownGenes table (downloaded September 10, 2012).Gene expression data in FPKM were generated using cufflinks 2.1.1 and pre-processed by collapsing on 27,979 unique gene symbols (sum of FPKM values of each matching transcript), keeping 18,802 NCBI RefSeq NM-category (mRNA) gene symbols and adding to each expression measurement 0.1 FPKM, performing a log2 transformation.PAM50 subtyping was performed using an implementation of the Parker method (Parker et al., J.Clin Oncol 2009). In short, to avoid context dependency when assigning PAM50 subtype by nearest-centroid, a fixed reference was selected to match the original cohort used by Parker et al. with respect to available clinical characteristics. Before subtyping tumors in this study, gene expression of the PAM50 genes for each tumor was centered to the reference set separately using custom R scripts.Genome_build: Human genome reference GRCh37/hg19 (with b37 masked chromosome Y and hs37d5 decoy sequences).Supplementary_files_format_and_content: FPKM gene expression in CSV format. | | Submission date | May 17, 2016 | | Last update date | Mar 12, 2018 | | Contact name | Lao H Saal | | E-mail(s) | [email protected] | | Organization name | Lund University | | Department | Department of Oncology and Pathology | | Lab | Translational Oncogenomics Unit | | Street address | Scheelevägen 2, MV404B2 | | City | Lund | | ZIP/Postal code | 22391 | | Country | Sweden | | Platform ID | GPL11154 | | Series (2) | | GSE81538 | Clinical Value of RNA Sequencing–Based Classifiers for Prediction of the Five Conventional Breast Cancer Biomarkers: A Report From the Population-Based Multicenter Sweden Cancerome Analysis Network—Breast Initiative [cohort 405] | | GSE81540 | Clinical Value of RNA Sequencing–Based Classifiers for Prediction of the Five Conventional Breast Cancer Biomarkers: A Report From the Population-Based Multicenter Sweden Cancerome Analysis Network—Breast Initiative [superseries] |
| | Relations | | BioSample | SAMN05006268 |
| Supplementary data files not provided | | Raw data not provided for this record | | Processed data are available on Series record |
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