title: TBS of Homo sapiens: unexposed male PBMC
design: The EZ DNA Methylation-Gold™ Kit (Zymo Research, USA) was used for the bisulfite conversion of DNA samples. Bisulfite treatment of samples was done according to the manufacturer's instructions with some changes at incubation and elution stages to reduce salts in final bisulfite-converted DNA samples (BS-DNA) and degradation of DNA. The input of DNA was between 400-500 ng and quantification of BS-DNA was done by spectrophotometer with RNA settings. The regions of interest in the ESR1, IGF2, H19, APOA5, NFKB1 and CDKN2A genes were selected based on sequences from UCSC genome browser (Genome Reference Consortium Human Build 38) and literature review. By design, targets were enriched for differentially methylated regulatory regions and TF binding sites, which predominantly carry H3K27ac and H3K4me3 annotations, mainly located in promoters, CpG Islands. The primer design for target locations was performed using BS-DNA as a template with the online tool BiSearch. The product sizes were analyzed by the MultiNA microchip electrophoresis system to exclude non-specific amplification. Further, 54 pairs of bisulfite-specific primers were selected and used for target enrichment applying to the 48.48 Access Array Integrated Fluidic Circuit (Fluidigm, USA). In total 68 indexed amplicons were synthesized for each sample and deep-sequenced.
library_name: DC_19