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Sample GSM1662765

Query DataSets for GSM1662765

Status

Public on Oct 30, 2015

Title

Blood_Platelets_Panc-448

Sample type

SRA

Source name

448-Pancr-KRAS

Organism

Homo sapiens

Characteristics

tissue: blood
cell type: Thrombocytes
patient id: Panc-448
cancer type: Pancreas
batch: Batch03
mutational subclass: KRAS

Extracted molecule

total RNA

Extraction protocol

Blood platelets were isolated from whole blood in purple-cap BD Vacutainers containing the EDTA anti-coagulant. The cells and aggregates were removed by centrifugation at room temperature for 20 minutes at 120g, resulting in platelet-rich plasma. The platelets were isolated from the platelet-rich plasma by centrifugation at room temperature for 20 minutes at 360g. The platelet pellet was collected in 30 μl RNAlater (Life Technologies), incubated overnight at 4°C and frozen at -80°C for further use. Frozen platelets were thawed on ice and total RNA was isolated using the mirVana RNA isolation kit (Life Technologies) according to the manufacturers’ protocol. Complementary purification of small RNAs was included in the isolation procedure by addition of miRNA homogenate (Life Technologies). Total RNA was dissolved in 30 μl Elution Buffer (Life Technologies) and RNA quality and quantity was measured using Bioanalyzer 2100 with RNA 6000 Picochip (Agilent).
100-500 pg of platelet total RNA (Bioanalyzer RIN values >7 and/or distinctive rRNA curves) diluted in nuclease free H2O was subjected to cDNA synthesis and amplification using the SMARTer Ultra Low RNA Kit for Illumina Sequencing v1 (Clontech, cat. nr. 634936) according to the manufacturers’ protocol. Conversion and efficient amplification of cDNA was quality-controlled using the Bioanalyzer 2100 with DNA High Sensitivity chip (Agilent). Samples with detectable fragments in the 300-7500 bp region were selected for further processing and Covaris shearing by sonication (Covaris Inc). Sample preparation for Illumina sequencing was performed using the Truseq DNA Sample Preparation Kit (Illumina, cat nr. FC-121-2001) or Truseq Nano DNA Sample Preparation Kit (Illumina, cat nr. FC-121-4001). Sample quality and quantity was measured using the DNA 7500 chip or DNA High Sensitivity chip (Agilent). High-quality samples with product sizes between 300-500 bp were pooled in equimolar concentrations (8-12 samples per Hiseq lane) and submitted for 100 bp Single Read sequencing on the Hiseq 2500 platform (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Single-end 100 bp reads were subjected to pre-aligment 5'-end quality trimming and sequence adapter clipping using Trimmomatic.
Cleaned-up reads were mapped to the hg19 reference genome using STAR (version 2.3.0), using splice aware alignments and allowing for 10 mismatches.
Selection of intron-spanning reads was performed using Picard-tools (version 1.115)
Read summarization was performed using HTSeq (version 0.6.1) using union-mode, unstranded reads and minimal mapping quality of 35, all guided by the Ensembl gene annotation version 75.
Genome_build: hg19
Supplementary_files_format_and_content: Samples in the tab-delimited text file were processed according to the described protocol and data of all samples was merged in a comprehensive read count matrix.

Submission date

Apr 21, 2015

Last update date

May 15, 2019

Contact name

Myron Best

E-mail(s)

m.best@vumc.nl

Organization name

VU University Medical Center

Department

Neurosurgery