Homo sapiens
Protocols: Thymocytes were isolated by Ficoll-Hypaque (GE Healthcare) from the cell suspensions obtained by thymic tissue manual dispersion. CD4SP Tregs and Tconvs were sort-purified based on the surface expression of CD4, CD8, CD27, CD25 and CD127 in FACS Aria III (BD Biosciences), reaching purities always above 95%. CD3 was intentionally not used to avoid possible signalling, but the sorting strategy was always further validated in parallel using CD3 and intracellular Foxp3 in a Fortessa flow cytometer (BD Biosciences). The staining protocols have been previously described (Nunes-Cabaço et al., 2010). Thymic samples were obtained during paediatric reconstructive cardiac surgery, using tissue that would be otherwise discarded from children without evidence of immunodeficiency or syndromatic diseases. ATAC-seq was performed following the Omni-ATAC protocol (Corces et al., 2017) with minor modifications. 5x104 sorted tTreg or tTconv cells were lysed for 3 minutes on ice, in 50uL of ATAC-Resuspension Buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2) containing 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. tn5 tagmentation was performed using TDE1 Enzyme and Buffer TD (Illumina) at 37ºC for 30 minutes, shaking at 1000rpm. After purification with a MinElute PCR Purification Kit (Qiagen), samples were amplified with NEBNext High Fidelity 2x PCR Master Mix (New England Biolabs) with index adapters from (Buenrostro et al., 2015): i5- AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTCAGATGTG i7- CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTCTCGTGGGCTCGGAGATGT (barcodes identified as NNNNNNNN). Final PCR reaction was then purified with a MinElute PCR Purification Kit followed by size-selection (150bp-1000bp using Ampure XP beads (Beckman Coulter).
replicate
3
disease
normal
individual
T353
organism
Homo sapiens
organism
Homo sapiens
sex
male
scientific_name
Homo sapiens
common name
human
organism part
thymus
developmental stage
infant
isolate
not applicable
ENA-FIRST-PUBLIC
2023-11-11T01:23:43Z
ENA-LAST-UPDATE
2023-11-23T11:54:38Z
cell_type
CD4-positive, CD25-positive, alpha-beta regulatory T cell
age
1
ENA-FIRST-PUBLIC
2023-11-11
ENA-LAST-UPDATE
2023-11-23