GSM6395350: MethylSeq_CAD9_T0; Homo sapiens; Bisulfite-Seq
from whole blood sample (300µl) using FlexiGene DNA Kit (QIAGEN) according to manufacturer instructions. SeqCap Epi Enrichment Kit ROCHE + Epi CpGiant capture ROCHE library prep, bisulfite, capture Genomic DNA has been manually extracted from whole blood samples (300µl) using FlexiGene DNA Kit (QIAGEN) according to the manufacturer's instructions. The concentration of the isolated DNA was evaluated by using a NanoDrop ND-1000 Spectrophotometer (Euro-Clone).  Then, to converts all unmethylated cytosines to uracil, bisuphite treatment was performed on genomic DNA (2 µg/µl) using EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) according to the manufacturer's instructions. The genomic DNA treated was quantified by the Qubit®2.0 Fluorometer (ThermoFisher Scientific, Massachusetts, USA).   Total RNA extraction from PBMCs was performed using Trizol® (Ambion, Waltham, MA, USA) reagent according to manufacturer instructions. RNA concentration was evaluated by using a NanoDrop ND-1000 Spectrophotometer (Euro-Clone), whereas RNA quality was checked on agarose gel 1%. To isolate miRNAs fraction, 50ng of RNA has been reverse transcribed into cDNA using the miScript II RT kit (QIAGEN) following the manufacturer's instruction. Genome-wide methylation study: for library preparation, Kapa ROCHE together with CpGiant ROCHE Nimblegen has been used. MiRNA sequencing: sequencing libraries were generated using SMARTer small RNA CLONTECH for Illumina following the manufacturer's recommendations (New England BioLabs, Ipswich, MA, USA). Genome-wide methylation and miRNA-sequencing has been performed using NextSeq 500 ILLUMINA platform. All samples passed quality control analysis.