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| Status |
Public on Dec 15, 2017 |
| Title |
C57BL/6J_Female_NaiveCD4+Tcell_A5 |
| Sample type |
SRA |
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| Source name |
from CD4+CD62L+ T cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: spleen
cell type: CD4+CD62L+ T cells
gender: female
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| Growth protocol |
C57BL/6 mice at 6-8 weeks of age were used for spleen collection purposes.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Naïve CD4+ T lymphocytes were purified from single-cell suspensions by MACS depletion of non-CD4+ T cells as well as regulatory T cells and TCRγ/δ+ T cells, using a cocktail of specific biotin conjugated antibodies against CD8a (Ly-2), CD45R (B220), CD49b (DX5), CD11b (Mac-1), Ter-119, CD25 and TCRγ/δ+ using the CD4+ CD62L+ T Cell Isolation Kit II (Miltenyi) to obtain an unlabeled pre-enriched CD4+ T cell fraction. Collected cells were suspended in PBS containing 2% FBS and Fc receptors were blocked with FcR blocking reagent for 10 minutes at 4°C (Miltenyi). Cells were further fluorescently stained with a combination of CD4-FITC (Miltenyi), CD44-APC clone IM7 (eBioscience), CD62L clone MEL-14 (BD Pharmingen), eFluor® 450 CD25 clone PC61.5 (eBioscience), for 10 minutes at 4°C, washed and then stained with DAPI (Life Technologies) to cell sort the live population of naïve CD4+, CD62Lhi, CD44lo, CD25- T lymphocytes using the MoFlo XDP system (Beckman Coulter). Samples were gated on single cells for doublet discrimination and non-viable cells were excluded using the viability dye 4’, 6 – diamidine – 2 – phenylindole (DAPI). AllPrep DNA/RNA Mini Kit (Qiagen) was used to perform simultaneous isolation of RNA and DNA from 10^6 sorted naïve CD4+ T cells and resting B cells. All RNA samples were subjected to in-column RNase-Free DNase treatment (Qiagen) to remove traces of DNA contamination. RNA integrity was subsequently assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies) by using the RNA 6000 Pico Kit (Agilent Technologies). For each sample type 3 biological replicates were generated from male and female samples.
RNA was extracted from 1 million cells and spiked-in with synthetic ERCC ExFold RNA Spike-In Mixes (Ambion®) according to manufacturer’s instructions. RNA-sequencing libraries were prepared at the Wellcome Trust Sanger Institute using the bespoke pipeline and the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (Illumina) following manufacturer’s instructions with minor modifications. 400ng of RNA per sample type were subjected to rRNA-depletion with RiboZero Gold, fragmented for 6 min using metal-ion catalysed hydrolysis. Following the adaptor ligation step an Uracil-Specific Excision enzymatic treatment (NEB) was incorporated to introduce nicks in U positions of the second strand and ensure optimal stranded library preparation. The libraries were amplified with 8 cycles of PCR using the KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems), cleaned up with AMPure XP Beads, quantified and pooled in equimolar amounts. The pools were sequenced at the Wellcome Trust Sanger Institute on the Illumina HiSeq 2000 platform using 100bp paired-end read length.
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| Library strategy |
RNA-Seq |
| Library source |
genomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
C57BL/6J_Female_NaiveCD4+Tcell_A5
T_B_F_1_A5
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| Data processing |
trim_galore -o $outputfile --fastqc --gzip --paired --stringency 3 $fastq1 $fastq2
For alignment we used the STAR aligner (Dobin,2012). star --runThreadN 4 --genomeDir $genome_dir --readFilesIn $fastq1 $fastq2 --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 10 --outFilterMismatchNmax 999 –outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --readFilesCommand zcat --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM GeneCounts --limitBAMsortRAM 40000000000 --chimSegmentMin 10 --alignSoftClipAtReferenceEnds No
We produced unstranded coverage files using: STAR --runThreadN 4 --runMode inputAlignmentsFromBAM --inputBAMfile Aligned.sortedByCoord.out.bam --outWigType bedGraph --outWigStrand Unstranded --outWigReferencesPrefix chr
To quantify gene expression we used the isolator framework (Jones,2016) isolator analyze --threads 1 -g /data/genomes/mm10/mm10-ercc.fa --output isolator-output.hdf5 gencode.vM3.annotation.gtf C57BL6-isolator.yaml > out ; We then use isolator summarize gene-expression --credible=0.95 --normalize isolator-output.hdf5 to produce estimates for gene expression.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Feb 08, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolas Walker |
| Organization name |
University of Cambridge
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| Department |
Genetics
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| Lab |
Ferguson-Smith
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| Street address |
Downing Street
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| City |
Cambridge |
| ZIP/Postal code |
CB2 3EH |
| Country |
United Kingdom |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE94671 |
The BLUEPRINT Murine Lymphocyte Epigenome Reference Resource [RNA-seq] |
| GSE94676 |
The BLUEPRINT Murine Lymphocyte Epigenome Reference Resource |
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| Relations |
| BioSample |
SAMN06312170 |
| SRA |
SRX2544815 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2480731_T_B_F_1_A5-signal-unstr.bw |
261.9 Mb |
(ftp)(http) |
BW |
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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