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Sample GSM1194689

Query DataSets for GSM1194689

Status

Public on Jul 03, 2014

Title

Patient 3 premalignant 2

Sample type

SRA

Source name

Premalignant lesion

Organism

Homo sapiens

Characteristics

patient: Patient 3
pathology: Premalignant
tissue: Premalignant lesion

Extracted molecule

total RNA

Extraction protocol

Tissues were sectioned at a 7-micron thickness and mounted on regular uncharged glass slides for patients 1, 2, and 3, and on polyethylene napthalate (PEN) membrane slides (Leica) for patient 4, followed by H&E staining. LCM was performed using the Arcturus eIIx for patient 1 and 2, Zeiss PALM for patient 3, and Leica LMD7000 for patient 4. A tissue area of 800,000 to 1,200,000 μm2 was dissected and collected from each lesion. RNA was extracted from laser-microdissected cells using the RNeasy Micro Kit (QIAGEN).
The cDNA was generated using the Ovation RNA-Seq System (NuGEN) for patients 1 and 2 and the 15 Ovation RNA-Seq V2 System (NuGEN) for patients 3 and 4. For patients 1 and 2, cDNA of ~200 bp was selected by gel purification. For patients 3 and 4, the cDNA was sheared to 140-180 bp using the Covaris focused-ultrasonicator with the following settings: duty cycle 10%; intensity 5; cycles per burst 200; total time 6 minutes.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Laser-microdissected premalignant lesion

Data processing

Single-end sequencing was performed for 36 cycles (patients 1 and 2) or 50 cycles (patients 3 and 4). Basecalling was performed for all cycles for patients 1, 3 and 4 and for the first 35 cycles for patient 2. Illumina CASAVA software version 1.6.0 (patients 1 and 2) or 1.7.0 (patients 3 and 4) was used for basecalling. Reads that failed Illumina's chastity filter [brightest intensity / (brightest intensity + second brightest intensity) < 0.6 for at least two of the first 25 cycles] were automatically removed during preprocessing.
After chastity filtering, the first 35 bases of each read were aligned to the human genome (build hg19) using Bowtie v0.12.7 (the first base of reads from patient 1 was omitted during alignment due to a technical issue). Only unique alignments were allowed, with up to two mismatches per read, with the command: 'bowtie -p 2 -a --best --strata -v 2 -m 1 -S --fullref -t'
Coverage estimates were computed by merging exons from all isoforms of each of 55,841 Ensembl Gene (ENSG) loci (Ensembl build 69) to create a single meta-transcript for each ENSG, and counting the number of reads falling in the exons of each meta-transcript using the BEDTools software suite.
Reads aligning to the mitochondrial genome were removed from further analysis. Reads per kilobase per million aligned reads (RPKM) were computed for each remaining ENSG in each sample by normalizing counts to the size of each meta-transcript and to the number of reads aligned to the nuclear genome in each sample. RPKM values were seventh-root-transformed prior to analysis to produce an approximately normal distribution of (nonzero) gene expression values.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text file including the number of reads (counts) overlapping each gene and sample.
Supplementary_files_format_and_content: Tab-delimited text file including RPKM values for each gene and sample.

Submission date

Jul 23, 2013

Last update date

May 15, 2019

Contact name

Adam C Gower

E-mail(s)

agower@bu.edu

Phone

617-358-7138

Organization name

Boston University School of Medicine