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Status |
Public on Jan 01, 2017 |
Title |
Wild type activated Tregs rep2 |
Sample type |
SRA |
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Source name |
Spleen and lymph node
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Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 tissue: Spleen and lymph node cell type: Regulatory T cells (Treg) genotype: wild type activated
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Extracted molecule |
total RNA |
Extraction protocol |
Treg cells were sorted from pooled spleens and peripheral lymph nodes (not including the mesLN) of healthy adult and age matched Myb+/+FoxP3YFP-Cre/YFP-Cre wild type and Mybfl/flFoxP3YFP-Cre/+ mosaic females. Cell suspensions were depleted of CD8 (53-6.7) and CD19 (1D3)-biotin labeled cells using streptavidin magnetic microbeads (Miltenyi), following the manufacturer instructions. Cells were incubated with anti-FcγR for 10 min on ice, stained with CD4-pacific blue, TCRβ-APC, CD62L-PE, ICOS-PECy7, TIGIT-PerCPeFluor710 and Sytox Blue. Sorted cells were collected in PBS with 50% FCS, washed twice in PBS, resuspended in RLT buffer (Qiagen) with β-Mercaptoethanol (Sigma), and kept at -80ºC. RNA was isolated independently from two biological replicates (each replicate being a pool of up to 4 independent sorts) with RNeasyPlus Mini kit (Qiagen). mRNA reverse transcription and cDNA libraries were prepared using the TruSeq RNA Sample preparation kit (Illumina) following the manufacturer’s instructions.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NextSeq 500 |
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Data processing |
Sequence reads were aligned to the GRCm38/mm10 build of the Mus musculus genome using the Subread aligner. Only uniquely mapped reads were retained. Genewise counts were obtained using featureCounts. Reads overlapping exons in annotation build 38.1 of NCBI RefSeq database were included. Immunoglobulin genes, T-cell receptor genes and genes located on Y chromosome were excluded from analysis. Genes were filtered from downstream analysis if they failed to achieve a CPM (counts per million mapped reads) value of 0.5 or greater in at least two libraries. Counts were converted to log2 counts per million, quantile normalized and precision weighted with the ‘voom’ function of the limma package. A linear model was fitted to each gene, and empirical Bayes moderated t-statistics were used to assess differences in expression. Empirical Bayes moderated-t P values were adjusted to control the global false discovery rate (FDR) across all comparisons with the ‘global’ option of the limma package. Genes were called differentially expressed if they achieved a FDR of 0.05 or less. Genome_build: mm10 Supplementary_files_format_and_content: tab-delimited text files include normalized log2-RPKM values for genes. Raw counts are available on the series record.
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Submission date |
Aug 28, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Wei Shi |
E-mail(s) |
Wei.Shi2@monash.edu
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Organization name |
Monash University
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Street address |
Wellington Rd
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City |
Clayton |
State/province |
Victoria |
ZIP/Postal code |
3800 |
Country |
Australia |
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Platform ID |
GPL19057 |
Series (1) |
GSE72494 |
Effector regulatory T cell differentiation and immune homeostasis depend on the transcription factor Myb |
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Relations |
BioSample |
SAMN04017589 |
SRA |
SRX1167445 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1863260_WT_act_rep2.txt.gz |
178.7 Kb |
(ftp)(http) |
TXT |
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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