|
| Status |
Public on Dec 13, 2016 |
| Title |
Control_3 |
| Sample type |
SRA |
| |
|
| Source name |
RNA from CD4+ T Cells isolated from the spleen
|
| Organism |
Mus musculus |
| Characteristics |
treatment: control
|
| Treatment protocol |
Parent animals were treated with 0.5 g/l of each Vancomycin, Streptomycin, Ampicillin and Metronidazole in the drinking water containing 4 g/l Splenda. Offspring mice were weaned at day 21 and kept on sterile drinking water until analysis. Control animals were conventionally raised (without antibiotics), weaned at day 21 and kept on regular drinking water until analysis.
|
| Growth protocol |
Cells were preenriched from the spleens of Foxp3 reporter mice on C57BL/6 background using the StemCell CD4 T cell isolation kit and sorted for CD4+ Foxp3- CD25- CD45Rbhi cells. RNA was isolated using the Qiagen Rneasy mini kit according to the manufacturer's instructions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA of CD4 T cells was extracted using the RNeasy Mini Kit from QIAGEN, following the manufacturer’s instructions.
RNA samples were quantified by qubit (Life Technologies) and quality by Agilent Bioanalyzer. All samples had RIN above 8. Libraries were prepared using TruSeq Stranded mRNA kit (Illumina). One hundred fifty nanograms from 6 RNA samples were purified for polyA tail containing mRNA molecules using poly-T oligo attached magnetic beads. Following purification, the RNA was fragmented. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” based was added and adapter ligated followed by purification and enriched with PCR to create cDNA libraries. Final cDNA libraries were size validated using Agilent Bioanalyzer and concentration validated by qPCR. All libraries were normalized to 10nM and pooled together. 10pM of pooled libraries were loaded onto Illumina cBot for cluster generation. Clustered flow cell was then sequenced Pair-end 100 cycles V3 using Illumina HighSeq 2000 to achieve a minimum of ~35 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
RNA-Seq of untreated CD4+ T Cells
|
| Data processing |
Raw data aligned using tophat2
Transcript abundance estimated using Cufflinks
Significantly differentially expressed genes calculated using cummeRbund
Genome_build: mm9
Supplementary_files_format_and_content: Results.csv comma separated value file. Details significantly differentially expressed genes between both conditions
Supplementary_files_format_and_content: AllGenesFPKM.csv comma separated value file. FPKM data for each condition for each gene. AllGenesFPKM.csv contains gene names.
|
| |
|
| Submission date |
Dec 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander James Murison |
| E-mail(s) |
alexander.murison@uhnresearch.ca
|
| Organization name |
University Health Network
|
| Lab |
Dick Lab
|
| Street address |
101 College Street, TMDT room 11-706
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1L7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13112 |
| Series (1) |
| GSE92282 |
Early life antibiotic treatment enhances the pathogenicity of CD4+ T cells curing intestinal inflammation |
|
| Relations |
| BioSample |
SAMN06132510 |
| SRA |
SRX2416299 |
