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Sample GSM1863261 Query DataSets for GSM1863261
Status Public on Jan 01, 2017
Title Wild type effector Tregs rep1
Sample type SRA
 
Source name Spleen and lymph node
Organism Mus musculus
Characteristics strain: C57BL/6
tissue: Spleen and lymph node
cell type: Regulatory T cells (Treg)
genotype: wild type effector
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
 
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.
 
Submission date Aug 28, 2015
Last update date May 15, 2019
Contact name Wei Shi
E-mail(s) Wei.Shi2@monash.edu
Organization name Monash University
Street address Wellington Rd
City Clayton
State/province Victoria
ZIP/Postal code 3800
Country Australia
 
Platform ID GPL19057
Series (1)
GSE72494 Effector regulatory T cell differentiation and immune homeostasis depend on the transcription factor Myb
Relations
BioSample SAMN04017590
SRA SRX1167446

Supplementary file Size Download File type/resource
GSM1863261_WT_effector_rep1.txt.gz 178.6 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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