title: GSM2255728: FNY01_3_3_MethylSeq; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA was extracted according to standard protcol Genomic DNA was extracted from about 50 million basophilic erythroblasts and libraries were produced using a protocol developed at the Einstein epigenomic facility. Extracted genomic DNA was fragmented (400-500 bp) with Covaris, end-repaired, dA tailed and then premethylated adapters (Illumina TruSeq adapters) were ligated at the ends of the fragmented DNA. The adapter ligated DNA samples were purified with AMPure XP beads (1:1 dilution) to eliminate adapter dimers and products with short insert, and then treated with sodium bisulphite using EZ DNA Methylation lightning kit (Zymo Research). The bisulphite treated products were used as a template for PCR amplification using following condition: 25 µl of 2x KAPA HiFi HotStart Uracil+ ReadyMix, 1.5 µl of 10 µM of primer P5, 1.5 µl of 10 µM of primer P7 and bisulphite treated library in a final volume of 50 µl; 98oC for 2 minutes, then 10 cycles of 98 oC for 30 seconds, 60 oC for 30 seconds and 72 oC for 4 minutes followed by 10 minutes at 72 oC for final extension. Amplified libraries were purified with MinElute PCR purification kit, and then we performed size selection with MinElute Gel Extraction kit (the insert size was 250 bp to 850 bp). For each of the two individuals analyzed, paired-end 2x100bp reads were then generated on six lanes of an Illumina HiSeq 2500. Methyl Seq library