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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 19, 2017 |
| Title |
WT 11weeks RNA-Seq replicate 3 |
| Sample type |
SRA |
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| Source name |
Synovial Fibroblasts
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| Organism |
Mus musculus |
| Characteristics |
cell type: Synovial Fibroblasts
passages: 4-5
strain: CBA x C57BL/6J
genotype: wild type
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from mouse SFs using the Absolutely RNA Miniprep Kit (Agilent Technologies) and from human SFs using the miRNeasy Mini kit (Qiagen). DNA was extracted from mouse SFs using the PureLink Genomic DNA Mini Kit (Invitrogen). In order to enrich for methylated DNA, the MethylMiner DNA Enrichment Kit from Invitrogen was used as per the manufacturer’s instructions. DNA was firstly fragmented by sonication to an average size of 250 bp. Then enrichment was performed with a double elution (high and low salt) in order to capture both sparsely- and densely-methylated DNA; fractions were combined in the end. To enrich for H3K4me3, DNA was processed using the iDeal ChIP-seq Kit (Diagenode) in combination with a ChIP-grade H3K4me3 antibody (Diagenode), as per the manufacturer’s instructions. Chromatin was firstly sheared by sonication using a Bioruptor (Diagenode) and then subjected to the H3K4me3 enrichment protocol with a minor modification: after performing an RNase I digestion for 30 min at 37oC, the de-crosslinked DNA was eluted using the QIAquick PCR Purification Kit (Qiagen). Enrichment (in comparison to a non-enriched input control) was confirmed by quantitative PCR.
All library preparations, next generation sequencing and quality control steps were performed by the McGill University and Genome Quebec Innovation Centre. More specifically, for RNA-seq, TruSeq RNA libraries were prepared and samples were run in an Illumina HiSeq2000 platform using a 100 bp paired-end setup. For MethylCap-seq, TruSeq genomic DNA libraries were prepared and samples were run in an Illumina HiSeq platform using a 100 bp paired-end setup. For ChIP-seq, TruSeq genomic DNA libraries were prepared and samples were run in an Illumina HiSeq platform using a 100 bp paired-end setup.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Processed/RNA-Seq/11weeks.diff
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| Data processing |
RNA-seq samples were analysed with the tophat-cufflinks pipeline (Trapnell C et al., Nat Protoc 2012, Kim D et al., Genome Biol 2013, Trapnell C et al., Bioinformatics 2009)
Reads were mapped to the mm9 genome with tophat2. Transcripts were assembled using cufflinks. Final transcriptome assembly was performed with cuffmerge and differential expression was identified with cuffdiff.
MethylCap- and H3K4me3-seq reads were mapped using the BWA tool (Li H et al., Bioinformatics 2009)
Differential Methylated and H3K4me3 marked regions were extracted with the use of diffReps (Shen L et al., PLoS One 2013)
The default normalisation procedure of diffReps and windows of 500 bases for MethylCap-seq and 1000 bases for H3K4me3-seq were used for the analyses.
Genome_build: mm9
Supplementary_files_format_and_content: Processed/RNA-Seq/3weeks.diff is the differential expression analysis output of cuffdiff. It includes all the results of the Tg197/WT RNA-Seq comparison(3 weeks).
Supplementary_files_format_and_content: Processed/RNA-Seq/8weeks.diff is the differential expression analysis output of cuffdiff. It includes all the results of the Tg197/WT RNA-Seq comparison(8 weeks).
Supplementary_files_format_and_content: Processed/RNA-Seq/11weeks.diff is the differential expression analysis output of cuffdiff. It includes all the results of the Tg197/WT RNA-Seq comparison(11 weeks).
Supplementary_files_format_and_content: Processed/RNA-Seq/8weekstnf.diff is the differential expression analysis output of cuffdiff. It includes all the results of the WTplusTNF/WT RNA-Seq comparison(8 weeks).
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| Submission date |
Feb 23, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
George Kollias |
| E-mail(s) |
kollias@fleming.gr
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| Organization name |
BSRC "Alexander Fleming"/National and Kapodistrian University of Athens
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| Department |
Institute of Immunology/Department of Physiology, School of Medicine
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| Street address |
34 Fleming Street
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| City |
Vari |
| ZIP/Postal code |
16672 |
| Country |
Greece |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE95251 |
Genomic responses of mouse synovial fibroblasts during TNF-driven arthritogenesis greatly mimic those of human rheumatoid arthritis (RNA-Seq). |
| GSE95256 |
Genomic responses of mouse synovial fibroblasts during TNF-driven arthritogenesis greatly mimic those of human rheumatoid arthritis |
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| Relations |
| BioSample |
SAMN06434289 |
| SRA |
SRX2583621 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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