Blood tissue genomic dna extraction kit

The genomic DNA was extracted from the FACS-sorted live cells by using the Blood & Tissue Genomic DNA Extraction kit (Qiagen, USA). For isolation of DNA from cells fixed and stained with anti-Foxp3 mAb, we used the protocol described previously (Hansmann et al., 2010 (link); Piper et al., 2014 (link)). Briefly, sorted cells (CD4⁺CD8⁻Foxp3⁺CD25⁺) were incubated with 300 μl lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 50 mM EDTA, 0.5% SDS, 0.1 μg/ml proteinase K, and 20 μg/ml RNase A) for 24 hours at 60°C. Then DNA was extracted by phenol/chloroform/isoamyl alcohol solution (25:24:1) and precipitated overnight by ethanol. Extracted genomic DNAs were converted by the EZ DNA methylation gold kit (Zymo Research, USA). Anti-sense (Ohkura et al., 2012 (link)) strands of bisulfite-treated DNA were then subjected to PCR for amplification of CNS2 (12 CpG motifs were analyzed). The PCR products obtained were cloned into the pGemT-easy vector (Promega, USA) and 10–30 individual clones from each sample were sequenced with M13 reverse primer (GAAACAGCT ATGACCATG).