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| Status |
Public on Jan 08, 2024 |
| Title |
Th17_replicate.1 |
| Sample type |
SRA |
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| Source name |
blood
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| Organism |
Mus musculus |
| Characteristics |
tissue: blood
cell line: lymphocytes
cell type: Th17
genotype: WT FoxP3-GFP reporter
treatment: in vitro polarized
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| Growth protocol |
CD4+ T cells were isolated from male Foxp3-GFP reporter mice and naïve T cells were cell sorted and activated with anti-CD3/CD28 under different polarizing conditions: Th0 (neutral), Th1, Th2, Th17, Treg as well as Th17s differentiated without TGFb (Th17-noTGFβ) representing an intermediate cell phenotype. Briefly, murine CD4+ T cells were purified using the MACS CD4+ T cell negative selection kit (Miltenyi Biotec) and naïve CD4+ T cells from WT, Foxp3-GFP were then sorted on the basis of a CD4+CD8-CD62L+CD44−GFP−CD25- expression profile on a FACSAria flow cytometer (BD Biosciences). T cell activation was performed 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, 1% penicillin/streptomycin (Gibco-Life technologies) and b-mercaptoethanol (50 μM). Cells were split 3 days later with medium supplemented with rhIL-2 (100U/mL). Cells were maintained in a standard tissue culture incubator containing atmospheric O2 and 5% CO2. Data from Tregs included in this study was previously published.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was extracted from 1x10^6 CD4 Th cells/ replicate using the Qiagen DNA mini kit according to manufacturer's instructions (Qiagen Hilden, Germany).
DNA was sheared using g-tubes (Covaris, USA) to ~10KB before library preparation with ligation sequencing kit (ONT Cat #SQK-LSK109) followed by whole genome sequencing on Nanopore MinION flow cells with pore 9.4.1 chemistry for 72h.
Libraries were enriched using Cas9-targeting approach for a panel of 10 genes.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
MinION |
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| Description |
Run1_barcode04
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| Data processing |
*library strategy: Single molecule DNA methylation WGS
Raw read quality was determined with pycoQC
Raw fast5 files were converted into pod5 format and basecalled with ‘dorado’ (version 0.4.2, ONT), allowing the simultaneous delineation of 5mCpG and 5hmCpG using the high accuracy model (‘[email protected]’, ONT) for the detection of modified bases. Dorado was also used for simultaneous alignment to the mm10 assembly and demultiplexing, followed by sorting and indexing with ‘samtools’ (version 1.13). ‘Modkit’ (version 0.2.2) was used for piling of methylation data into bed files.
Bed files containing 5mC and 5hmC information were directly used for comparison after loading in R.
Assembly: mm10
Supplementary files format and content: bed files with methylation pileup data for each demultiplexed sample
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| Submission date |
Nov 24, 2023 |
| Last update date |
Jan 09, 2024 |
| 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) |
| GSE248540 |
Single molecule DNA methylation reveals unique epigenetic identity profiles of T helper cells |
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| Relations |
| BioSample |
SAMN38410082 |
| SRA |
SRX22633047 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7916547_Run1_barcode04.bed.gz |
6.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
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