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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 15, 2022 |
| Title |
WT_CD4_SP_BR1 |
| Sample type |
SRA |
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| Source name |
Thymus
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
cell subset: CD4+HSAlowTCRb+
Sex: female
genotype: wild type
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| Treatment protocol |
Mice were euthanized by CO2 asphyxiation and thymi were isolated and dissociated to acquire single-cell suspensions. Total thymocytes were depleted of CD24hi cells by staining with biotinylated mouse anti-CD24 (clone:M1/69) purchased from Biolegend. Unwanted cells were depleted by binding to mouse streptavidin magnetic beads (anti-mouse Rapidspheres, cat no 19860, Stemcell Technologies) according to the manufacturer’s instructions. Enriched cells were stained with viability dye (eBioscience) to distinguish live cells from. Cells were stained with aGalactosyl-Ceramide loaded tetramer (conjugated with PE, obtained from NIH tetramer core), TCRb (clone:H57-597) conjugated with PERCP/Cy5.5 (Biolegend), anti-mouse CD4 AF488 (clone:RM4-5), CD8 (clone:53-6.7) conjugated with Brilliant Violet 650 or APC. Live, CD24 low enriched cells CD4+, TCRb+, aGalCerCd1d tetramer-, CD8- cells were sorted in a purity higher than 98% and were used for RNA isolation.
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| Growth protocol |
All mice were bred and maintained under specific pathogen-free conditions at University of North Carolina (UNC) Genetic Medicine building, in a facility managed by the Division of Comparative Medicine at UNC Chapel Hill. The experimental procedures were approved by the UNC Institutional Animal Care and Use Committee.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Live, CD24 low enriched cells CD4+, TCRb+, aGalCerCd1d tetramer-, CD8- cells were sorted in a purity higher than 98%. RNA was isolated using the micro RNeasy plus kit (Qiagen
RNA was quantified using HS RNA kit (Invitrogen) in a Qubit 4 Fluorometer (Invitrogen). RNA integrity was assessed in a Bionalyzer (Agilent) using RNA 6000 pico kit (Agilent, cat no: 5067-1513). The RNA that was used for downstream steps had a RIN value of 9 or higher. Libraries were prepared using the SMARTseq kit v4 Ultra Low Input RNA kit for sequencing (Clontech, cat no: 634888) and Nextera XT (Illumina)
RNA-seq (SMART-seq)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
bcl2fastq ver 2.20.0
Adapter trimming and quality filtering of the sequencing libraries was done using fastp (0.21.0).
The sequencing libraries were mapped against mm10 and the GRCm38.100 transcriptome using STAR (2.7.5a).
The differential expression analysis was done using DESeq2 based on the read counts per gene produced by STAR.
The TCR repertoire extraction was done using MiXCR (3.0.13) from RNA-seq data using the “-p rna-seq” mode
Genome_build: mm10
Supplementary_files_format_and_content: STAR produced gene read counts and MiXCR produced clone count information.
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| Submission date |
Dec 06, 2021 |
| Last update date |
Jul 15, 2022 |
| Contact name |
Tarmo Äijö |
| Organization name |
Flatiron Institute
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| Department |
Center for Computational Biology
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| Street address |
162 5th Avenue
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| City |
New York |
| ZIP/Postal code |
10010 |
| Country |
USA |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE190230 |
TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner [RNA-Seq] |
| GSE206450 |
TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner. |
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| Relations |
| BioSample |
SAMN23673954 |
| SRA |
SRX13323497 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5718501_TSAGLB_01-WT_CD4_SP_BR1_CGTACTAG-AGAGTAGA_S1_L008ReadsPerGene.out.tab.gz |
297.1 Kb |
(ftp)(http) |
TAB |
| GSM5718501_clones_TSAGLB_01-WT_CD4_SP_BR1_CGTACTAG-AGAGTAGA_S1_L008.TRA.txt.gz |
450.3 Kb |
(ftp)(http) |
TXT |
| GSM5718501_clones_TSAGLB_01-WT_CD4_SP_BR1_CGTACTAG-AGAGTAGA_S1_L008.TRB.txt.gz |
516.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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