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| Status |
Public on Aug 17, 2016 |
| Title |
High_fat_diet_germ_cell_smallRNA_rep4 |
| Sample type |
SRA |
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| Source name |
High fat diet, germ cell
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| Organism |
Rattus norvegicus |
| Characteristics |
cell type: germ cell
strain: Sprague-Dawley
gender: male
diet: High fat diet
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| Treatment protocol |
Rats were fed from weaning on control diet (10% calories from fat) (D06072701, Research diets) or high fat diet (40% calories from fat) (D06071701, Research Diets) until 19 weeks of age. Testes were decapsulated and minced with scalpels in 5 ml ice cold Hanks Buffered Saline Solution with calcium and magnesium and supplemented with 0.1% Collagenase IV (Sigma, UK). The suspension was mechanically dissociated by pipetting before incubation at 37°C with gentle rotation for 10 minutes. The suspension was then passed through a 40µm cell strainer (Becton Dickinson and Company, UK) which was further washed with 10 ml of phosphate buffered saline (PBS) supplemented with 2% FCS (Invitrogen, UK). The cells were washed by centrifugation at 500xg for 5 mins and resuspended in 10ml 2% FCS three times. Samples were kept on ice until FACS on a BD Aria II, gating for expression of eGFP.
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| Extracted molecule |
total RNA |
| Extraction protocol |
smallRNA libraries were assembled using the Truseq small RNA kit (Illumima) according to the maunfacturers instructions. smallRNAs of 22-10nt in length were selected for sequencing.
6x10e6 sorted cells were then centrifuged and RNA immediately extracted using the Qiagen miRNAeasy mini kit (Qiagen, UK) according to the manufacturer’s instructions. Quality and purity of the extracted total RNA was verified using spectrophotometry and the Agilent RNA 6000 nano kit according to manufacturer’s instructions
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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 |
HFD_GC_R4_smallRNA
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| Data processing |
RNAseq reads were aligned to rn5 using Star v2.3.0, with a custom splice junction database constructed from Ensembl v73 protein-coding gene models (--outFilterMismatchNoverLmax 0.05)
smallRNAseq reads were trimmed with trimmomatic to remove adaptor contamination to a minimum length of 22 nucleotides(MINLEN:22)
Trimmed smallRNAseq reads were iteratively aligned to rn5 using Butter (perl wrapper for bowtie designed to iteratively map small RNA seq reads to maximize read assignment to the genome).
Reads were counted over ensembl v73 protein-coding gene models (collapsed models where multiple transcripts exist) and repBase repeats for aligned RNAseq data using featureCounts. Aligned smallRNA seq data were counted over miRBase, piRNAQuest annotations using featureCounts
Genome_build: rn5
Supplementary_files_format_and_content: .tsv.gz files contain integer read counts over genome annotations from featureCounts; tab delimited.
Each file contains 7 columns, with rows representing a single aggregated feature. For instance, an aggregate feature may be all of the exons of a gene, or transcribed repeat elements of the same class/type that are indistinguishable at multiple locations.
The columns (in order) are:
* Geneid - annotation ID for the aggregated features
* Chr - ";" separated list of genome contigs for all component features
* Start - ";" separated list of contig start positions for all component features
* End - ";" separated list of contig end positions for all component features
* Strand - ";" separated list of strand annotation for all component features
* Length - total genomic length of aggregated features
* <samplefilename> - number of read counts over aggregated features
For more details on the featureCounts software see the user guide:
http://bioinf.wehi.edu.au/subread-package/SubreadUsersGuide.pdf
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| Submission date |
Apr 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Morgan |
| E-mail(s) |
michael.morgan@ndcls.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Department of Physiology, Anatomy and Genetics
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| Lab |
CGAT
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| Street address |
South Parks Road
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| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX1 3PT |
| Country |
United Kingdom |
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| Platform ID |
GPL18694 |
| Series (1) |
| GSE80721 |
Effect of high fat diet on the rat germ cell transcriptome |
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| Relations |
| BioSample |
SAMN04909040 |
| SRA |
SRX1733508 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2134290_Rn_germ-HFD_smRNA-2327_12.butter_vs_repeats.tsv.gz |
28.2 Mb |
(ftp)(http) |
TSV |
| GSM2134290_Rn_germ-HFD_smRNA-2327_12.butter_vs_rn5_mirbase.tsv.gz |
15.5 Kb |
(ftp)(http) |
TSV |
| GSM2134290_Rn_germ-HFD_smRNA-2327_12.butter_vs_rn5_pirna.tsv.gz |
7.6 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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