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Sample GSM833108

Query DataSets for GSM833108

Status

Public on Jan 25, 2012

Title

Sample3Reduced

Sample type

SRA

Source name

globin reduced blood

Organism

Homo sapiens

Characteristics

tissue: whole blood

Treatment protocol

From each sample, 1.5 µg of RNA was treated using the GLOBINclear™ Human Kit (Ambion, Austin, TX, USA) according the manufacturer’s instructions. The kit contains 10 globin sequence specific biotinylated oligonucleotides (http://www.freepatentsonline.com/y2006/0257902.html). Transcripts hybridized to the oligonucleotides are removed through streptavidin magnetic beads. Concentration and quality of the samples were checked as described above.

Growth protocol

Samples from six healthy individuals of Caucasian decent (4 females, 2 males) aged from 39 to 70 years old were collected after informed consent and with ethical approval. Whole blood was drawn into PAX gene tubes (Qiagen, Venlo, The Netherlands) and inverted 10 times. Samples were allowed to equilibrate at room temperature for 2 hours, placed at -20oC overnight and stored at -80oC until further processing. Before total RNA extraction, samples were thawed overnight at 4oC and total RNA was isolated using the PAX RNA isolation kit following the manufacturer’s instructions, including DNAse treatment. RNA quality and the RIN values were determined using the RNA Nano LabChip assay (Agilent Technologies, Santa Clara, CA, USA). The concentration of each sample was validated using the Nanodrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)

Extracted molecule

total RNA

Extraction protocol

SAGE libraries were produced according to the Illumina protocol for digital gene expression tag profilling with Nlaii. In short, after hybridization of 1μg total RNA to polydT magnetic beads (Dynabeads, Invitrogen Life Technologies, Carlsbad, CA, USA), first and second strand synthesis was performed. The beads attached to the double stranded DNA were digested with NlaIII restriction endonuclease to produce short double stranded constructs starting at the most 3’ CATG of the transcript. After ligation of the GEX adapter 1, the construct was digested with MmeI to create a 21 base pair fragment downstream of the GEX adapter 1. This fragment was then ligated to GEX adapter 2 to complete the cassette. The adaptor ligated constructs were amplified by 15 cycles of PCR and the products were loaded on 6% Novex TBE precast acrylamide gels (Invitrogen). The 96 base band corresponding to the NlaIII construct was excised and purified from the gel slice using the soak and crush method followed by ethanol precipitation. Sample quality was checked on a DNA 1000 LabChip (Agilent). Sequencing was performed at the Leiden Genome Technology Center on Illumina GA2 sequencer (Illumina, San Diego, CA, USA). Purified samples were diluted to 10nM and loaded on a single lane of the flow cell where, after cluster amplification, samples were put through an ultra short 18 cycle sequencing run.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

other

Instrument model

Illumina Genome Analyzer IIx

Data processing

The FASTQ files were analysed using the open source GAPSS_B(v2) pipeline (www.lgtc.nl\GAPSS). All sequences were trimmed to 17 base pairs to remove the first lower quality base pair from the 3' end of the sequences. After trimming, the NlaIII recognition site (CATG) was added to the 5' end of the sequence to create the complete 21-22mer nucleotide sequences. Sequences were aligned using the Bowtie short read aligner (version 0.12.7) against the UCSC hg19 reference genome, allowing for a maximum of one mismatch and a maximum of two possible positions in the genome (options: -k 1 -m 2 -n 1 --best --strata –solexa1.3-quals).A custom Perl script was used to create reference region files from the SAGE region files that were composed of the overlapping tags from all samples. A second Perl script was then used to link all individual region files to the reference region file, reporting the number of tags in each individual region of the reference region file. Finally, the reference region file was annotated with transcript information using BIOMART (Ensembl build 60). For all reported downstream statistical and biological analysis only sequences aligned to known exons were used. Analyses were performed at the gene level, and in case of multiple SAGE tags per gene, e.g. as a consequence of alternative polyadenylation, tags were summed.

Submission date

Nov 15, 2011

Last update date

May 15, 2019

Contact name

Anastasios Mastrokolias

Phone

0031715269425

Organization name

Leiden University Medical Center

Street address

LUMC, Building 2 Einthovenweg 20 2333 ZC Leiden