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Status |
Public on Jul 20, 2015 |
Title |
HiSeq_E4 |
Sample type |
SRA |
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Source name |
Whole-blood
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Organism |
Homo sapiens |
Characteristics |
tissue: Whole-blood input amount (rna): 1 ug purification method: AMPure XP beads rin value: 7.9
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Extracted molecule |
total RNA |
Extraction protocol |
Peripheral blood samples were collected in PAXgene blood RNA tubes (PreAnalytix, Switzerland). PAXgene tubes were frozen using a sequential freezing process. This involves storing tubes at room temperature for 3h, transferring to 4°C overnight, followed by 6-8h at -20°C and then final storage at -80°C. Total RNA (including the miRNA fraction) was isolated from whole-blood using the PAXgene Blood miRNA Kit (Qiagen, Canada), according to manufacturer's instructions. Furthermore, total RNA was isolated from frozen brain, heart and liver tissues using the miRNeasy Mini Kit protocol (Qiagen, Canada) with no modifications. RNA and miRNA yield and quality were determined using the Nanodrop 1000 (Thermo Scientific, USA) and Agilent 2100 Bioanalizer (Agilent Technologies, USA). All libraries were prepared using the Illumina TruSeq Small RNA protocol following the manufacturer's instructions with 12 cycles of PCR amplification after ligation of the 3' and 5' adapters. Individual libraries were prepared using a unique index primer in order to allow for pooling of multiple samples prior to sequencing. Libraries were validated and quantified using an Agilent 2100 Bioanalyzer High Sensitivity DNA chip and qRT-PCR with the KAPA library quantification kit (Kapa Biosystems, USA). Sample C1 (control - human brain) was not sequenced. All additional samples (A1-A10), as well as sample AC (control - no purification method), were sequenced. Purification Method: In order to compare three different small RNA library purification methods, we prepared 11 libraries starting with 1µg of good quality total RNA (RIN>8). All libraries were prepared using total RNA extracted from peripheral blood of a single individual. The RNA was split into 11 aliquots and each was used as a technical replicate. In addition, we used total RNA from commercially available human brain as a library preparation control. Libraries were purified as follows: 1) Purification by Novex TBE PAGE gel: 50µl of amplified cDNA from samples A1-A3 and C1 were loaded into a 6% Novex gel and run for 80 minutes at 130-135 V. After cleaning the gel with RNase free water, a band was manually cut to contain all fragments sized 145-160nt, corresponding to mature miRNAs and other regulatory small RNA molecules; 2) Purification by Pippin Prep automated gel system (Sage 3%): The Pippin Prep system (PPS) allows automatic selection of specified cDNA products. 25µl of amplified cDNA from samples A4-A7 were loaded into a Pippin Prep machine. Furthermore, in order to test variability between machines, samples A4 and A5 were loaded into PPS1, while samples A6 and A7 were loaded into PPS2. Size selection was automated for products between 125 and 180nt; 3) Purification by AMPure XP beads: Biotinylated magnetic AMPure beads allow for selection of specified cDNA products bound to streptavidin. 50µl of amplified cDNA from samples A8-A10 were mixed and purified two times with AMPure XP beads at a 1.8:1 ratio (beads:sample). This ratio allows for optimal selection of all products higher than 100nt.
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Library strategy |
ncRNA-Seq |
Library source |
transcriptomic |
Library selection |
size fractionation |
Instrument model |
Illumina HiSeq 2500 |
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Description |
processed_data: ncRNA_Expression.txt
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Data processing |
All sequencing data were processed using CASAVA 1.8+ and extracted from FASTQ files Fastx_toolkit was used to trim the Illumina adapter sequences Additional filtering based on defined cutoffs was applied in order to obtain high quality data. These filters included: 1) Phred quality (Q) score higher than 30, 2) reads between 15-40nt in length, 3) adapter detection based on perfect-10nt match, and 4) removal of reads without detected adapter We used Bowtie to align reads to the human genome (GRCh37) We used ncPRO-seq in combination with miRBase (V20) to match them to known miRNA sequences We used the Rfam and NCBI's piRNA databases to map other small RNA sequences All sequencing data was normalized with the Bioconductor - DESeq2 package, using a detection threshold of 1 count per miRNA (present at least once in each of the libraries tested) Genome_build: Human genome (GRCh37) Supplementary_files_format_and_content: Tab-delimited text files include small RNA count values for each sample
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Submission date |
Jun 12, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Juan Pablo Lopez |
E-mail(s) |
juan.lopez@mail.mcgill.ca
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Phone |
514 7616131
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Organization name |
McGill University
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Department |
Human Genetics
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Lab |
McGill Group for Suicide Studies (MGSS)
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Street address |
6875 La Salle Blvd
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City |
Montreal |
State/province |
Quebec |
ZIP/Postal code |
H4H1R3 |
Country |
Canada |
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Platform ID |
GPL16791 |
Series (1) |
GSE69825 |
Biomarker discovery: Quantification of microRNAs and other small non-coding RNAs using next generation sequencing |
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Relations |
BioSample |
SAMN03771404 |
SRA |
SRX1057759 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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