GSM5009375: MII-12m-4-D; Mus musculus; Bisulfite-Seq
Bisulfite-seq: To construct the scBS-seq libraries, 60fg of unmethylated lambda DNA was added into the retained lysis solution containing a nucleus. Pico Methyl-Seq Library Prep Kit (Zymo) was used for library construction strictly followed by the manufacturer's manual. The quality of all constructed libraries was assessed by Agilent Bioanalyzer 2100 with High Sensitivity DNA Kit (Agilent Technologies). Qualified libraries were sequenced on Illumina HiSeq4000 to generate 150bp pair-end reads." RNA and gDNA were separated from a single oocyte followed by the published protocol with minor modification. Frozen oocytes in PCR tubes were thawed at room temperature for around 5 minutes. The 7µl of soft lysis buffer (500mM KCl, 100 mM Tris-HCl pH=8, 1.35mM MgCl2, 4.5mM DTT, 0.45% Nonidet P-40, 0.18U SUPERase-In and 0.36U RNase-inhibitor) was added into each PCR tube containing a single oocyte, and the lysate was inhibited at 4˚C for 30 min. The tubes were vortexed at room temperature for one minute and then were centrifuged at 1000xg for 5 min. RNA in the cytoplasm was released to the lysate and gDNA storing in the nucleus was at the bottom of the tube. The 6.5µl of lysate supernatant was carefully transferred to another PCR tube for the following RNA-seq. The remaining nucleus with around 2.5µl of lysis buffer was subjected for performing bisulfite sequencing.