GSM5009366: MII-12m-1-R; Mus musculus; RNA-Seq
RNA-seq: Library preparation for scRNA-seq was according to the modified protocol of Smart-seq2. In brief, the mixture of 2µl of 10mM oligo-dT30VN primer (5'-AAGCAGTGGTATCAACGCAGAGTACT30VN-3') and 2µl of 10mM dNTP mix was added into the lysate supernatant, and the sample was inhibited at 72˚C for 3 min. Then, the 9.5µl of reverse transcription mixture (200U SuperScript II reverse transcriptase, 20U RNAse inhibitor, 1x Superscript II first-strand buffer, 5mM DTT, 1M Betaine, 6mM MgCl2, 1µM TSO (5'-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3') was added into the tube for RNA reverse transcription. The tubes containing total 20µl mixture were put into ProFlex PCR System (Thermo Fisher Scientific) and the reaction condition was as follows: 42˚C for 90min, followed by ten cycles of 50 ˚C for 2 min and 42 ˚C for 2 min. The final enzyme inactivation was performed at 70˚C for 15 min. After finishing the first-strand reaction, the 30µl of PCR preamplification mix (1x KAPA HiFi HotStart ReadyMix, 0.1µM IS PCR primers (5'-AAGCAGTGGTATCAACGCAGAGT-3')) were added into the tube containing 20µl of the first-strand reaction. PCR preamplification reaction was performed in ProFlex PCR System by using the following program: 98˚C for 3 min, 18 cycles of 98˚C for 20s, 67˚C for 15s and 72˚C for 6min, with the final extension cycle of 72˚C for 5min. Amplified cDNA was purified by using Ampure XP beads (1:1 ratio) according to the manufacturer's manual and was eluted in 15µl of ddH2O in the end. To check the quality of the cDNA library, 1µl of amplified cDNA was removed to perform additional same PCR amplification reaction as mentioned above and PCR product was run on a 1% agarose gel at 100 Volts for 45min. The yield of amplified cDNA was measure by Qubit3.0 fluorometer. 5ng of amplified cDNA with good quality were used for library construction with TruePrepTM DNA Library Prep Kit V2 for Illumina (Vazyme) followed by the instruction. 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.