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| Status |
Public on Apr 03, 2020 |
| Title |
Th17_KO2 (RNA-seq) |
| Sample type |
SRA |
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| Source name |
spleen, lymph nodes
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
cell type: in vitro-generated Th17 cells
culture time: 3 days
genotype: NCOR1f/f Cd4Cre
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared from 3x10^6 viable WT and NCOR1f/f,Cd4Cre in vitro-generated Th17 cells with QIAshredder, RNaeasy Mini Kit and RNase-Free DNase Set (Qiagen) according to manufacturer's protocol.
RNA-seq libraries were prepared with TruSeq Stranded mRNA LT sample preparation kit (Illumina) using Sciclone and Zephyr liquid handling robotics (PerkinElmer). Library amounts were quantified using Qubit 2.0 Fluorometric Quantitation system (Life Technologies) and the size distribution was assessed using Experion Automated Electrophoresis System (Bio-Rad). For sequencing, libraries were pooled, diluted and sequenced on an Illumina HiSeq 3000 instrument using 50 bp single-read chemistry.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Illumina HiSeq Control Software (HCS) HD 3.4.0.38 was used for the data acquistion on the sequencing instrument.
Basecalling was performed with the Illumina Real-Time Analysis (RTA) software 2.7.7.
BCL to BAM file conversion and demultiplexing was performed with custom programs based on Picard tools (v2.18.11, http://broadinstitute.github.io/picard/).
The transcriptome analysis was performed using the Tuxedo suite. TopHat2 (v2.1.1, http://genomebiology.com/2013/14/4/R36/abstract) was supplied with reads passing vendor quality filtering (PF reads) and the Ensembl transcript set (Homo sapiens, e87, December 2016) as reference. Tophat2 spliced alignments were run independently for each replicate.
Cufflinks (v2.2.1, http://www.nature.com/nbt/journal/v31/n1/full/nbt.2450.html) was then used to assemble transcripts from spliced read alignments, again using the Ensembl e87 transcriptome as the reference, preventing de novo assembly of transcript models.
Transcriptome sets of all replicates for each sample group were combined with Cuffmerge.
Differential expression was assessed with Cuffdiff v2.2.1 (http://www.nature.com/nbt/journal/v28/n5/full/nbt.1621.html).
The Bioconductor cummeRbund (http://www.bioconductor.org/packages/release/bioc/html/cummeRbund.html) pckage was used in combination with custom R scripts to perform quality assessment and further refine analysis results.
Genome_build: GRCm38 (UCSC mm10)
Supplementary_files_format_and_content: tab-separated value files of FPKM estimates obtained from Cufflinks, enriched with Ensembl transcrit annotation.
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| Submission date |
Oct 16, 2019 |
| Last update date |
Apr 04, 2020 |
| Contact name |
Wilfried Ellmeier |
| E-mail(s) |
wilfried.ellmeier@meduniwien.ac.at
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| Organization name |
Medical University of Vienna
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| Department |
Institute of Immunology
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| Street address |
Lazarettgase 19
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE138933 |
RNA-seq analysis of WT and NCOR1f/f,Cd4Cre in vitro-generated Th17 cells |
| GSE138934 |
Role of NCOR1 in CD4+ T cells |
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| Relations |
| BioSample |
SAMN13038863 |
| SRA |
SRX7004459 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4123856_Th17_KO2_S43989_genes_fpkm.tsv.gz |
1.7 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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