title: Nascent_Bis_Rep2 (CRX904662; CRA013943)
library_construction_protocol: Mouse Dhfr mRNA was served as methylation conversion control and 200ng mRNA mixed with Dhfr mRNA at a ratio of 300:1, which was fragmented into 100nt fragments for 1min at 90 centigrade using 10X RNA fragmentation reagent (Ambion) and stopped with 10X RNA stop solution (Ambion). After ethanol precipitation, the RNA was treated in 100microl bisulfite solution (pH 5.1) and incubated for 1 hour at 75 centigrade. The reaction mixture was then desalted by centrifugation through Nanosep with 3K Omega 500/pk columns (PALL Corporation). The RNA was washed with nuclease-free water for 5 times and dissolved in 100microl nuclease-free water following incubation with 100 microl 1M Tris-HCl (pH 9.0) for 1 hour at 75 centigrade. Precipitated with ethanol, the RNA was dissolved and reverse transcribed using superscript II Reverse Transcriptase (Invitrogen) and ACT random hexamers.