GSM4998045: young oocyte rep1; Mus musculus; Bisulfite-Seq
Before scBS seq, 2 × lysis buffer and 0.5 μ lproteinase K were added followed by incubation at 37 C for 1 h. For hippocampus WGBS, 200-300mg tissue were used, genome DNA concentration per sample were all >= 50 ng/μl.The A260/A280 ratio of gDNA was 1.8~2.0 and the electropherogram of gDNA was without RNA protein and degradation. For WGBS of hippocanpus,a total volume of 80 μl gDNA unmethylated lambda DNA were combined and fragment the DNA to 300bp.Then the next protocol include End repair, dA Tailing, Methylated a dapt o r ligation, and bisulfite conversion. The uracil binding pocket of KAPA HiFi DNA Polymerase has been inactivat was then used in PCR amplification, enabling amplification of uracil containing DNA . For scBs seq, before incubation at 65 C for 3 min ,50 U of Klenow exo (Sigma) were added and the samples incubated at 4 C for 5 min, 37 C for 30 min. Samples were incubated at 95 C  for 1 min and transferred immediately to ice before addition of fresh oligo 1 (10 pmol), Klenow exo (25 U), and dNTPs (1 nmol) in 2.5 μ l total. The samples were incubated at 4 C for 5 min , 37 C for 30 min. This ran dom priming and extension was repeated a further three times (five rounds in total). Samples were then incubated with 40 U exonuclease I (NEB) for 1 h at 37 C before DNA was purified using 0 .8 × Agencourt Ampure XP beads.Samples were eluted in 10 mM TrisCl (pH 8.5) and incubated with washed M 280 Streptavidin Dynabeads (Life Technologies) for 20 min with rotation at room temperature. Beads were washed twice  and resuspended in dNTPs, 0.4 μ M oligo 2 and 1 × Blue Buffer. Samples were  incubated at 95 C for 45 s and transferred immediately to ice before addi tion of 100 U Klenow  exo (Sigma) and incubation at 4 C for 5 min, +1 C/15 s to 37 C, 37 C for 90 min.Libraries were then amplified by PCR as follows: 95 2 min, 12 13 repeats of (94 C 80 s, 65 C 30 s, 72 C 30 s), 72 C 3 min and 4 C hold. Whole Genome Bisulfite Sequencing (WGBS)