GSM5212544: melan-a UVR 4; Mus musculus; OTHER
Cells were pelleted and lysed in 2% SDS, 25 mM EDTA; proteins were precipitated by the addition of 10M ammonium acetate and removed by centrifugation. DNA was precipitated by the addition of isopropanol and washed twice with 70% ethanol. The DNA pellet was dissolved in TE (Tris 10 mM, EDTA 1mM). High molecular DNA was recovered by immobilization on 0.4x AMPure XP beads (Beckman Coulter) prior to methylation analysis. Two micrograms of genomic DNA were spiked with methylation standards and then sequentially digested with SmaI and XmaI restriction enzymes. DNA was digested with 20 units of SmaI for 8 h at 25°C. 20 units of XmaI was added and the digestion was continued for an additional 16 h at 37°C. 3' end filling and dA tailing was performed by 15 units of Klenow Fragment (3'>5' exonuclease deficient; NEB) with nucleotide mix (0.4 mM final concentration of each dCTP, dGTP, dATP). Libraries were prepared by ligation of NEBNext adapters  at a 10:1 adapter to fragment ratio using T4 DNA ligase (NEB) at 16°C overnight. Hairpin loops of the adapters were cleaved with USER enzyme at 37°C for 15 min. Ligated DNA was dual-size selected using AMPure XP beads for fragments between 250 to 450 bp. Adapter-ligated DNA fragments were indexed with NEBNext Index  i7 primers (NEB). Libraries were amplified for 14 cycles and cleaned with AMPure XP beads prior to sequencing. Libraries were were pooled to 10 nM and spiked with 25% phiX library to ensure sequence diversity. OTHER: DREAM