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| Status |
Public on Feb 22, 2017 |
| Title |
Serum_Pool_B. tRNA_dep_Beads |
| Sample type |
SRA |
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| Source name |
Serum, tRNA-halves depleted using beads
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| Organism |
Mus musculus |
| Characteristics |
strain/background: 129/SVj
disease status: Healthy
gender: Pooled male and female
tissue: Serum
trna-halves depleted: Beads
serum pool: B
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| Extracted molecule |
total RNA |
| Extraction protocol |
Healthy male and female 129/SVj mice, with ages varying between 2 to 9 months, were sedated using tribromoethanol at a dose of 250 mg/kg injected intraperitoneally. 500 μl to 1 ml of blood per mouse was collected in Safe-Lock micro test tubes (Eppendorf) by cardiac puncture using a 0.45 x 12 mm needle, after which mice were sacrificed by cervical dislocation. Clotted blood samples from healthy SVj mice were centrifuged at 1900 g for 10 min at 4 °C using a swinging bucket rotor. The supernatant, containing the serum, was collected and centrifuged again at 16000 g for 10 min at 4°C in a fixed angle rotor to pellet cellular nucleic acids attached to cell debris. The supernatant was collected and stored at -80 °C. For all serum samples, the degree of hemolysis was determined by measuring levels of free hemoglobin by spectral analysis. Absorbance peaks at 414, 541 and 576 nm are indicative of free hemoglobin.
Serum samples from 3 individual SVj mice were pooled and 200 μl aliquots of the serum pool were used as input for RNA isolation using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer's instructions. Total RNA was eluted in 12 μl of RNase-free water. After elution from the miRNeasy column, RNA were diluted to a final volume of 38 μl and distributed in 12 μl aliquots.
Small RNA sequencing libraries were prepared using the TruSeq Small RNA Library Preparation kit v2 (Illumina) according to manufacturer's instructions using 6 µl of RNA as input. After PCR amplification (15 cycles), quality of libraries was assessed using a high sensitivity DNA kit on a Bioanalyzer (Agilent) according to manufacturer's instructions. Size selection was performed using 3% agarose dye-free marker H cassettes on a Pippin Prep (Sage Science) following manufacturer's instructions with a specified collection size range of 125-153 bp. Libraries were further purified and concentrated by ethanol precipitation, resuspended in 10 μl of 10 mM Tris-HCl (pH=8.5) and quantified using qPCR. Based on the qPCR results, equimolar library pools were prepared, quality was assessed as described above and the library was further diluted to 4 nM using 10 mM Tris-HCl (pH=8.5).
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
NSQ_Run116_B_beads-33117149
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| Data processing |
BaseSpace: FASTQ Generation | Version: 1.0.0 was used for FASTQ generation.
Adaptor trimming was performed using Cutadapt v1.8.1. Reads shorter than 15 bp and those in which no adaptor was found were discarded.
For quality control, the FASTX-Toolkit (v0.0.14) was used. A minimum quality score of 20 in at least 80% of bases was applied as a cutoff.
The reads were mapped with Bowtie (v1.1.2) without allowing for mismatches.
Mapped reads were annotated by matching genomic coordinates of each read with genomic locations of miRNAs (obtained from miRBase, v20) and other small RNAs (obtained from UCSC (mouse: GRCm38/mm10) and Ensembl, v84).
Genome_build: miRBase v20; Ensembl v84; GRCm38/mm10
Supplementary_files_format_and_content: *miRs.txt: Tab-delimited text files represent the raw miRNA count table for each sample.
Supplementary_files_format_and_content: *contam.txt: Tab-delimited text files represent other small RNA counts for each sample. Columns: Sample, Read ID, Read counts, Annotation.
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| Submission date |
Oct 18, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alan Van Goethem |
| Organization name |
Ghent University
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| Street address |
De Pintelaan 185
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| City |
Ghent |
| ZIP/Postal code |
9000 |
| Country |
Belgium |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE88913 |
Small RNA sequencing of serum-derived RNA for the evaluation of 5' tRNA halves depletion protocols |
| GSE88915 |
Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples |
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| Relations |
| BioSample |
SAMN05919678 |
| SRA |
SRX2251001 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2354259_NSQ_Run116_B_beads_contam.txt.gz |
63.8 Kb |
(ftp)(http) |
TXT |
| GSM2354259_NSQ_Run116_B_beads_miRs.txt.gz |
3.3 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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