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Sample GSM1709681

Query DataSets for GSM1709681

Status

Public on Jul 20, 2015

Title

MiSeq_D5

Sample type

SRA

Source name

Whole-blood

Organism

Homo sapiens

Characteristics

tissue: Whole-blood
input amount (rna): 0.05 ug
purification method: AMPure XP beads
rin value: 9.1

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.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina MiSeq

Description

processed_data: MiSeq.txt

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

Submission date

Jun 12, 2015

Last update date

May 15, 2019

Contact name

Juan Pablo Lopez

E-mail(s)

juan.lopez@mail.mcgill.ca

Phone

514 7616131

Organization name

McGill University