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| Status |
Public on Jul 15, 2022 |
| Title |
Tet2/3 DKO CD4 SP IgG control |
| Sample type |
SRA |
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| Source name |
Thymus
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| Organism |
Mus musculus |
| Characteristics |
genetic background: C57BL/6J
cells: CD4+HSAlowTCRb+
treatment: untreated
antibody: IgG (Epicypher, cat no: 13-0042)
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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. For the thymic differentiation related studies, live CD4+, CD8-, TCRb+, tetramer - cells were sorted and usedfor CUT&RUN. The purity of the samples after sorting was >98%.
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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 |
genomic DNA |
| Extraction protocol |
500.000 CD4 single positive (SP) cells, sorted by Flow cytometry, were used as starting material and were processed using the CUT&RUN kit (Epicypher).
. The antibodies used for these experiments are: GATA3 (clone: D13C9) XP (Cell Signalling, cat no: 5852), IgG (Epicypher, cat no: 13-0042). 3ng DNA (corresponding to small fractions of DNA) were used for library preparation utilizing NEBNext Ultra II DNA Library Prep Kit (NEB). The adaptor was diluted to 1:12.5 for adaptor ligation. The adaptor-ligated fragments were amplified for 12 PCR cycles using a 10 sec 60 degrees Celsius annealing/extension step. After amplification, libraries were purified with 1.0x volume of AMPure XP beads (Beckman). The concentration of DNA was measured using the double stranded (ds) DNA High Sensitivity (HS) assay in a Qubit 4 Fluorometer (Invitrogen). The quality control and the size of the library was determined using High Sensitivity (HS) D1000 Screen Tape assay (Agilent, cat no: 5067-5584), including HS D1000 screen tape and HS D1000 reagents (such as HS D1000 sample buffer and HS D1000 ladder, Agilent, cat no: 5067-5585), in a Tapestation 4150 (Agilent).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Thymus_CD4_B92-2_IgG
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| Data processing |
Library strategy: CUT&RUN
bcl2fastq ver 2.20.0
Adapter trimming and quality filtering of the sequencing libraries was done using fastp (0.21.0) with the default parameters.
The sequencing libraries were mapped against mm10 using Bowtie 2 (2.4.1) (--very-sensitive -X 2000). Mitochondrial reads were removed after alignment. Additional filtering was done using samtools (1.12) using the following parameter values: -q 30 -h -b -F 1804 -f 2.
Reads with identical sequences were filtered and only one was retained for subsequent analysis.
The peaks were identified from the pooled samples against controls using HOMER (4.10) (findPeaks -style factor).
The coverage tracks were generated from the samples obtained by pooling the biological replicates using HOMER.
Genome_build: mm10
Supplementary_files_format_and_content: Coverage tracks (bigWig) produced by HOMER.
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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 |
GPL24247 |
| Series (2) |
| GSE190228 |
TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner [CUT&RUN] |
| GSE206450 |
TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner. |
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| Relations |
| BioSample |
SAMN23673942 |
| SRA |
SRX13323466 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5718482_Thymus_CD4_B92-2_IgG.ucsc.bigWig |
175.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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