GSM5229304: Young myoblast WGBS 3; Mus musculus; Bisulfite-Seq
Genomic DNA was extracted using phenol/chloroform/isoamyl alcohol solution (Invitrogen  #15593-031). Primary myoblasts were pelleted at 0.6xg for 10 minutes and snap frozen in liquid  nitrogen before use. When all samples were collected, cell pellets were thawed on ice and lysed  in 400ul lysis buffer (40nM Tris-HCl pH8.0; 1% (vol/vol) Triton X-100; 0.1% (vol/vol) SDS; 4  mM EDTA (pH 8.0) and 300 mM NaCl). Cell lysates were incubated at 37°C for 45 minutes  with 10ul RNase A (Invitrogen, cat#12091-021), followed by a 1h15min incubation at 45°C with  10ul of Proteinase K (10mg/ml Millipore, cat#70663). 1 volume of phenol/chloroform were then  added to each lysate, and samples were vortexed vigorously and centrifuged at maximum speed  for 3 minutes. The aqueous layer was retained and mixed with 2 volumes of 100% EtOH.  Samples were snap frozen in liquid nitrogen for 3 minutes, thawed on ice, and centrifuged for 20  minutes at maximum speed. The supernatant was discarded, and the DNA pellet was washed 3x  with 750ul of 75% EtOH, spinning each time at maximum speed for 12 minutes. The DNA was  then air-dried and resuspended in TE buffer. Genomic DNA was sheared using COVARIS, and libraries were constructed using the NxSeq AmpFREE low DNA Library Kit by Lucigen. Bisulfite conversion was done using the EZ-96 DNA Methylation-GoldTM MagPrep kit by Zymo Research.