Assay summary for BS-seq_Cairns_Lab_DNA Methylation_0001AS
Cairns Lab, HHMI
1-3ug of DNA was sheared on a COVARIS Adaptive Focused Acoustics S-series system (Woburn, Massachusetts 01801-1721) to a median fragment size of 400 bp. Sheared DNA for sperm, sphere stage and muscle were purified using QIAquick PCR purification kit (QIAGEN, Valencia, CA 91355), 100ng-1ug of sheared or digested DNA was used in making the library. For all samples unmethylated lambda DNA was added to 1% final concentration (Promega Madison, WI 53711) as a control for conversion efficiency. Methylated paired-end adapters were ligated to sheared DNA according to the Illumina library preparation protocol and purified using QIAquick PCR purification kit (QIAGEN, Valencia, CA 91355). Sodium bisulfite conversion was performed using the EpiTect Bisulfite Kit (QIAGEN, Valencia, CA 91355) with an additional incubation step at 60C for 120 min to increase conversion rate. Bisulfite treated DNA was then PCR ampli- fied with 16 cycles. Size distribution and quality was checked on Agilent DNA 1000 Bioanalyzer chip DNA Chip and qPCR using the KapaBiosystems KAPA Library Quant Kit. Samples were sequenced with either a 101-base paired-end or single-end format on Illumina HiSeq 2000.