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| Status |
Public on Feb 22, 2017 |
| Title |
NCr Mouse 1069. TimePoint 1.tRNA dep |
| Sample type |
SRA |
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| Source name |
Serum, before tumor, tRNA-halves depleted
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| Organism |
Mus musculus |
| Characteristics |
strain/background: NCr
gender: Female
treatment: None
tissue: Serum
trna-halves depleted: Yes
mouse id: NCr Mouse 1069
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| Growth protocol |
Orthotopic neuroblastoma xenografts were generated in four- to six-week-old female athymic immunodeficient mice. Briefly, one million human SH-SY5Y neuroblastoma cells were surgically implanted in beneath the renal capsule. Serum samples were collected before and two weeks after tumor cell injection.
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| Extracted molecule |
total RNA |
| Extraction protocol |
100 µl blood was collected by puncture of the jugular vein using a 4 mm lancet and collected in a BD Vacutainer collection tube with a gel separator (BD). All blood samples clotted at room temperature for 45 min. Clotted blood samples were centrifuged at 2000 g for 15 minutes at 4°C in a fixed angle rotor. 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.
RNA was isolated from 50 μl of serum that was collected from Ncr nude mice and eluted in 12 μl of RNase-free water using the miRNeasy serum/plasma kit (Qiagen) according to the manufacturer's instructions. For RNA samples that are depleted for tRNA-halves, biotinylated DNA probes complementary to the 5' tRNA-halves were allowed to hybridize to the target RNA sequences. Magnetic streptavidin beads were used to immobilize the DNA-RNA complexes. Using a magnetic field, the beads and bound DNA-RNA complexes were separated, after which the supernatant, containing tRNA-depleted RNA, was collected and purified using ethanol precipitation and suspended in 7.5 µl RNase-free water.
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 |
miRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
18270_dep-35014183
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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 (human: GRCh37/hg19; mouse: GRCm38/mm10) and Ensembl, v84).
Genome_build: miRBase v20; Ensembl v84; Human: GRCh37/hg19; mouse: GRCm38/mm10
Supplementary_files_format_and_content: *.txt: Tab-delimited text files represent the raw miRNA count table for each sample created by mapping either to the mouse genome or the human genome.
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| Submission date |
Oct 13, 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) |
| GSE88728 |
Small RNA sequencing of serum collected from mice before and after manifestation of tumor |
| GSE88915 |
Depletion of tRNA-halves enables effective small RNA sequencing of low-input murine serum samples |
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| Relations |
| BioSample |
SAMN05907903 |
| SRA |
SRX2245065 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2344865_18270_dep-35014183_miRs_human.txt.gz |
1.6 Kb |
(ftp)(http) |
TXT |
| GSM2344865_18270_dep-35014183_miRs_mouse.txt.gz |
2.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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