WGBS of mus musculus: E13.5 embryos
The sequencing libraries of 12 embryo and 1 oocyte samples were constructed as follows: 2 lysis buffer and 0.5 l proteinase K were added to gathered cells followed by incubation at 37C for 1 h. Bisulfite conversion was performed on cell lysates using an EZ DNA Methylation-Gold kit as well as WGBS. After purification, DNA was eluted in 10 mM Tris-Cl and combined with oligo 1. Before incubation at 65C for 3 min, 50 U of Klenow exo (Sigma-Aldrich) were added, and the samples were incubated at 4C for 5 min and at 37C for 30 min. Samples were incubated at 95C for 1 min and transferred immediately to ice before the addition of fresh oligo 1 (10 pmol), Klenow exo (25 U), and dNTPs (1 nmol) in a 2.5 l total. The samples were incubated at 4C for 5 min and at 37C for 30 min. The random priming and extension were repeated a further three times (five rounds in total). Samples were then incubated with 40 U exonuclease I (New England Biolabs) for 1 h at 37C before DNA was purified using 0.8 Agencourt Ampure XP beads (Beckman Coulter, Brea, CA, USA). Samples were eluted in 10 mM Tris-HCl (pH 8.5) and incubated with washed Streptavidin Dynabeads M-280 (Life Technologies, Carlsbad, CA, USA) for 20 min with rotation at room temperature. Beads were twice washed and resuspended in dNTPs, 0.4 M oligo 2 and 1 Blue Buffer. Samples were incubated at 95C for 45 s and transferred immediately to ice before addition of 100 U Klenow exo and incubation at 4C for 5 min, +1C/15 s to 37C, 37C for 90 min. Libraries were then PCR-amplified as follows: at 95C for 2 min, 1213 cycles of (at 94C for 80 s, at 65C for 30 s, at 72C for 30 s), at 72C for 3 min, and a 4C hold. Purified libraries were assessed for quality and quantity using Agilent Bioanalyzer and StepOnePlus Real-Time PCR System. Single cell libraries were prepared for 125-bp paired-end sequencing on a HiSeq 2500.