4 protocols
growth protocol
Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation from leukapheresis products of healthy volunteers. CD4+ T cells were enriched with the human CD4+ T Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). CD4+CD25highCD127low/- Treg cells were isolated by FACS (BD Biosciences, USA) from enriched CD4+ T cells and cultured in X-Vivo 15 medium (Lonza, USA) supplemented with 5% human AB serum (Sigma-Aldrich, USA). Cells were stimulated with anti-CD3/CD28 Abs-coated beads (Invitrogen-Dynal, USA) in the presence of 500 U/ml recombinant human IL-2 (PeproTech, USA) for 8 days and then rested in the presence of 100 U/ml IL-2 for 14 days. After two cycles of stimulation, cells were rested for 4-5 days.
nucleic acid library construction protocol
Total RNA was extacted using TriPure Reagent. RNA libraries were prepared for sequencing using standard Illumina protocols
sample treatment protocol
Expanded cells were sorted into FOXP3+ and FOXP3-losing cells by FACS (BD Biosciences, USA). For intracellular staining, washing and following cell sorting steps, the PBS with 0.1% diethyl pyrocarbonate (DEPC; Sigma-Aldrich, USA) was used. The buffer was autoclaved and supplemented with recombinant RNasin® Ribonuclease Inhibitor (Promega, USA) just before use.
nucleic acid library construction protocol
Cells were crosslinked with 1% formaldehyde and chromatin was sonicated to obtain an average fragment length of 200 to 500 bp. After preclearing with protein A agarose beads (Upstate, USA), sonicated chromatin was precipitated with anti-H3K4m3 and anti-H3K27m3 (abcam, United Kingdom) overnight at 4℃. The immune complexes were bound to protein A agarose beads (Upstate, USA). After washing and elution, crosslinks were reversed at 65℃ overnight. The eluted DNA was treated sequentially with Proteinase K and RNase A, and purified with the QIAquick PCR-purification kit (Qiagen, Germany). The enrichment efficiency of ChIP was detected using qPCR approach. The purified DNA fragments was repaired using PNK and Klenow enzyme, and ligated to adapters. Subsequently, PCR-amplified fragments of around 100-300bp were sequenced using Illumina HiSeq™ 2000 following manufacturer’s protocols (www.illumina.com).