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Sample GSM2344861 Query DataSets for GSM2344861
Status Public on Feb 22, 2017
Title NCr Mouse 1046. TimePoint 1.tRNA dep
Sample type SRA
 
Source name Serum, before tumor, tRNA-halves depleted
Organism Mus musculus
Characteristics strain/background: NCr
gender: Female
treatment: None
tissue: Serum
trna-halves depleted: Yes
mouse id: NCr Mouse 1046
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.
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).
 
Library strategy miRNA-Seq
Library source transcriptomic
Library selection size fractionation
Instrument model Illumina NextSeq 500
 
Description 18247_dep-35014177
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.
 
Submission date Oct 13, 2016
Last update date May 15, 2019
Contact name Alan Van Goethem
Organization name Ghent University
Street address De Pintelaan 185
City Ghent
ZIP/Postal code 9000
Country Belgium
 
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
Relations
BioSample SAMN05907895
SRA SRX2245061

Supplementary file Size Download File type/resource
GSM2344861_18247_dep-35014177_miRs_human.txt.gz 1.6 Kb (ftp)(http) TXT
GSM2344861_18247_dep-35014177_miRs_mouse.txt.gz 2.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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