GSM2335685: mut_vitc_wgbs; Mus musculus; Bisulfite-Seq
Bisulfite conversion was performed on genomic DNA using the MethylEdge Kit (Promega) according to manufacturer recommendations. To monitor under and over conversion rates 1ng of fully methylated T7 phage and unmethylated lambda DNA were spiked into 100ng of sample gDNA prior to bisulfite conversion. The resulting pool was suspended in 5ul of EB buffer, added to 32.5 μl of MethylEdge Bisulfite Conversion Reagent and incubated at 98°C for 6 min followed by 54°C for 60 min. Bisulfite converted DNA was isolated with MagSi-DNA all-around beads (MagNa Medics) and desulfonation was performed on the beads for 15 min before 2 washes 80% ethanol and elution for 15 min @ 56C. cDNA was generated from the resulting single stranded template by adding 2.5 μl of 10 mM dNTP mix (NEB) and 2 μl of 500 μM random primer to the DNA and denaturing at 98°C for 1 min before quickly moving to ice and adding 5 μl of 10X NEB2 buffer (NEB) and 1 μl of 50 U/μl Klenow fragment (3′→5′ exo−) (NEB). The mixture was then incubated as follows: 4°C for 30 min; 4°C-37°C (1°C/min); 37°C for 30 min; 70°C for 10 min. After cDNA synthesis the DNA was purified using SeraMag magnetic beads (20% PEG; 1M NaOH) a 75ul:50ul bead:DNA ratio, and in 35 μl before standard Illumina library preparation beginning at the adapter ligation stage. Genomic DNA/RNA was extracted using the Qiagen AllPrep Mini Kit according to the manufacturer's instructions.
GEO Accession
GSM2335685