title: GSM6048479: SW1353_RRBS_rep2_G30; Homo sapiens; Bisulfite-Seq
library_construction_protocol: Genomic DNA from ARPE-19,Jurkat and SW1353 was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's protocol.Two microgram of genomic DNA was used to construct the RRBS library by NEBNext Ultra II library kit and EZ DNA Methylation-Gold™ Kit (Zymo Research) following the manufacturer's protocol. The quality of libraries was assessed with Agilent 2100 Bioanalyzer (Agilent, United States). To evaluate the conversion error of bisulfite conversion, the methylated spike in and unmethylated spike in were used. We chose two different regions as the templates from the λDNA. One template was amplified with dNTP Mix to synthesize the unmethylated spike in, and the other one was amplified with 5-Methylcytosine dNTP Mix to synthesize the methylated spike in. Then the spike in were fragmented to about 300bp by Covaris (Covaris, USA). After constructing libraries using NEBNext Ultra II Library Prep Kit for Illumina® separately, the methylated spike in and unmethylated spike in were mixed together and spiked to the DNA at a ratio of 1% before bisulfite conversion. Two micrograms of genomic DNA were used for MspI (NEB, San Diego, California, USA) digestion. And the fragments were recovery by using AxyPrep PCR Clean kit. After constructing the sequencing library and mixing the spike in, bisulfite conversion was performed on the libraries by the EZ DNA Methylation-Gold Kit (Zymo Research, USA), which was also used to recovery the libraries. Epimark Hot Start Enzyme was used for PCR to amplify the libraries. To obtain the fragments exactly, the vertical TBE-PAGE electrophoresis was performed on the 6% TBE-PAGE Gel (Novex, Invitrogen, USA) to better separate the bands. After recovering the libraries from the gel, the Agilent 2100 Bioanalyzer system (Agilent, USA) was used for assessing the distribution of libraries.