title: GSM8879818: oocyte_PBAT_3; Mus musculus; Bisulfite-Seq
library_construction_protocol: Cell lysis, BS conversion and DNA purification were performed with the EZ DNA Methylation-Direct Kit (Zymo Research, D5021) following the manufacturer's instructions. Cell lysis, BS conversion and DNA purification were performed with the EZ DNA Methylation-Direct Kit (Zymo Research, D5021) following the manufacturer's instructions. The eluted DNA were mixed with 0.4 mM dNTPs (Takara, 4019), 0.4 μM oligo 1 (5'-Biotin-CTACACGACGCTCTTCCGATCTNNNNNNNNN-3') and 1x Blue Buffer (TIANGEN, NG202) (24 μl final) before incubation at 65 ℃ for 3 min followed by 4 ℃ pause. Then the samples were combined with 50 U of Klenow exo- (TIANGEN, NG202) and incubated at 4 ℃ for 5 min, + 1 ℃/15 s to 37 ℃, 37 ℃ for 1 h. Samples were incubated at 95 ℃ for 1 min and transferred to ice immediately before addition of fresh oligo 1 (10 pmol), Klenow exo- (25 U), and dNTPs (1 nmol) in 2.5 µl total. The samples were incubated at 4 ℃ for 5 min, + 1 ℃/15 s to 37 ℃, 37 ℃ for 1 h again. This random priming and extension step repeated 7 times in total. Samples were then incubated with 40 U exonuclease I (NEB) for 1 h at 37 ℃ and purified with 0.8x VAHTS DNA Clean Beads. Samples were eluted in 50 µl Elution Buffer (QIAGEN) and mixed with 50 µl washed M-280 Streptavidin Beads (ThermoFisher, 11205D) before incubation at room temperature for 30 min. Beads were washed twice with 0.1 M NaOH, twice with Wash Buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 2 M NaCl) and resuspended in 48 µl of 0.4 mM dNTPs, 0.4 µM oligo 2 (5'-AGACGTGTGCTCTTCCGATCTNNNNNNNNN-3') and 1x Blue Buffer. Samples were incubated at 95 ℃ for 45 s and transferred to ice immediately before addition of 100 U of Klenow exo- and incubated at 4 ℃ for 5 min, + 1 ℃/15 s to 37 ℃, 37 ℃ for 1.5 h. Then beads were washed twice with 50 µl Elution Buffer and resuspended in 50 µl Amplification mix including 25 µl 2x KAPA HiFi HotStart ReadyMix (Kapa Biosystems, KK2602), 23 µl Nuclease-Free Water (ThermoFisher, AM9932), 1 µl Universal PCR Primer and 1 µl Index PCR Primer (NEBNext Multiplex Oligos for Illumina (NEB, E7335S)). Libraries were then amplified by PCR as follows: 95 ℃ for 3 min, 12-15 repeats of (98 ℃ for 20 s, 60 ℃ for 30 s, 72 ℃ for 1 min), 72 ℃ for 5 min and 4 ℃ hold. Amplified libraries were purified using VAHTS DNA Clean Beads (Vazyme, N411) following the manufacturer's guidelines and sequenced on the Illumina NovaSeq 6000.