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| Status |
Public on Dec 08, 2021 |
| Title |
demultiplexed_Treg_assay_2 |
| Sample type |
SRA |
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| Source name |
Tregs
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| Organism |
Mus musculus |
| Characteristics |
assay: barcoded_multiplexed_cas9.enriched_assay_2
ligation kit: SQK-LSK109
barcoding kit: EXP-NBD104
barcodes: RB8
cell type: Treg
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| Treatment protocol |
When indicated, cell permeable αKG (dimethyl ketoglutarate, 3.5 mM; Sigma), malonate (dimethyl malonate, 10mM; Sigma) was added. Cells were split 2-3 days later with medium supplemented with rhIL-2 (100U/mL) and drugs were added at their original or half-dose concentrations. Cells were maintained in a standard tissue culture incubator containing atmospheric O2 and 5% CO2.
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| Growth protocol |
Murine CD4+ T cells were purified using the MACS CD4+ T cell negative selection kit (Miltenyi Biotec) and naive CD4+ T cells from female and male adult C57BL6/J, FoxP3-GFP, or ERBB2-CAR transgenic mice were sorted on the basis of a CD4+CD8-CD62L+CD44−GFP−CD25- expression profile on a FACSAria flow cytometer (BD Biosciences). Thymic Tregs (tTregs) were sorted from FoxP3-GFP reporter mice based on a CD4+CD8-CD62L+CD44−GFP+ profile. T cell activation (0.5-1e6 cells/well/ml) was performed on non-tissue-treated 24-well plates (ThermoFisher) using plate-bound α-CD3 (clone 145-2C11, 1 μg/ml) and α-CD28 (clone PV-1, 1 μg/ml) monoclonal antibodies in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS (PAN-Biotech, Eurobio), 1% penicillin/streptomycin (GIBCO-Life technologies) and β-mercaptoethanol (50 μM). For Treg-polarizing conditions, hTGF-β (3 ng/ml) and rhIL-2 (100 U/ml), were added to the cultures.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
T cells (from male mice) were polarized in the indicated conditions and DNA (3 μg) from each sample (2 biological replicates) was enriched in immune identity genes using cas9 guided RNPs, as previously published (Gilpatrick et al., 2020) and characterized (Goldsmith et al., 2021a; Goldsmith et al., 2021b).
TracrRNA and crRNA were purchased from Integrated DNA Technologies (IDT). Targeted genes included: Itgb8, Foxp3, Il17a, Rorc, Ifng, Il10, Maf, Il4 and Gata3. Samples were barcoded and multiplexed using the Nanopore Ligation Sequencing kit (SQK-LSK109) and Native barcode expansion kit according to manufacturer’s instructions (Oxford Nanopore Technology, Oxford UK) without PCR amplification.
Sequencing of native DNA was conducted on a MinION sequencer on ONT flow cells with protein pore R9.4 1D chemistry for 48h. MinION Mk 1B (ONT).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
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| Data processing |
Fast5 files were basecalled with Megalodon (using the Rerio ONT model “res_dna_r941_min_modbases_5mC_5hmC_v001.cfg”) and aligned with minimap2 to the mm10 mouse genome. The methylation status of each CpG site was determined by Megalodon after demultiplexing with Deepbinner (v.0.2.0) (Liu et al., 2021; Wick et al., 2018).
Genome_build: mm10
Supplementary_files_format_and_content: bed files (bedmethyl files, as described in megalodon documentation: https://nanoporetech.github.io/megalodon/)
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| Submission date |
Dec 07, 2021 |
| Last update date |
Dec 09, 2021 |
| Contact name |
Hector Hernandez-Vargas |
| E-mail(s) |
[email protected]
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| Organization name |
Genomics Consulting
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| Street address |
36 bis Rue de la Batterie
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| City |
Bron |
| State/province |
Rhone |
| ZIP/Postal code |
69500 |
| Country |
France |
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| Platform ID |
GPL24973 |
| Series (1) |
| GSE190409 |
Targeted nanopore DNA sequencing of mouse naive CD4 T cells activated under Treg polarizing conditions |
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| Relations |
| BioSample |
SAMN23765235 |
| SRA |
SRX13346255 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5723229_Treg_assay.2_modified_bases.5mC.bed.gz |
15.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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