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Sample GSM3475469

Query DataSets for GSM3475469

Status

Public on Nov 16, 2018

Title

86_spln_Id3pos_eTR [lib16890]

Sample type

SRA

Source name

spln_Id3pos_eTR

Organism

Mus musculus

Characteristics

mouse_id: 86
strain background: C57BL/6
genotype/variation: Id3-GFP x Foxp3-mRFP
tissue: Spleen
cell type: CD4+ T cells
cell population: Id3+CD62LloCD44hi effector TR (Id3pos_eTR)

Extracted molecule

polyA RNA

Extraction protocol

CD4+ T cells were isolated using CD4 microbreads (Miltenyi) from either peripheral LNs or spleens of 3 littermate Id3-GFP x Foxp3-mRFP mice. Cells were sorted based on viability, CD4, CD44, CD62L, Id3-GFP and Foxp3-mRFP expression on a FACs Aria II (BD Biosciences). 500 cells were sorted directly into SMART-Seq v4 Ultra Low Input RNA Kit (Takara) lysis buffer and protocol followed to produce cDNA.
Library construction was performed using a modified protocol of the NexteraXT DNA sample preparation kit (Illumina).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Base-calling was performed automatically by Illumina real time analysis software and demultiplexing was performed on Illumina BaseSpace.
Hard trimming was performed in Galaxy; 1 3'-end base was removed from all reads using the FASTQ Trimmer tool (v1.0.0)
Quality-based trimming was performed in Galaxy; reads were trimmed from both ends using the FASTQ Quality Trimmer tool (v1.0.0) until minimum base quality for each read was >= 30.
Reads were aligned in Galaxy using bowtie and TopHat (Tophat for Illumina tool, v.1.4.0)
Read counts per Ensembl gene ID were computed in Galaxy using htseq-count (htseq-count tool, v.0.4.1).
Sequencing, alignment, and quantitation metrics were obtained for FASTQ, BAM/SAM, and count files in Galaxy using FastQC, Picard, TopHat, Samtools, and htseq-count.
Genome_build: mouse NCBI genome build 37
Supplementary_files_format_and_content: raw _counts.txt: Read counts as quantified by htseq-count.
Supplementary_files_format_and_content: norm_counts.txt: Lowly expressed genes and non-protein coding genes were filtered out and the ensembl gene IDs mapped to MGI gene symbols. Count data was normalized using TMM (Robinson & Oshlack, 2010)

Submission date

Nov 15, 2018

Last update date

Nov 16, 2018

Contact name

Daniel J Campbell

Organization name

Benaroya Research Institute

Street address

1201 Ninth Avenue

City

Seattle