GSM4984597: 10-12month rep2; Mus musculus; RNA-Seq
The collected 2-cell embryos were washed twice with 1xPBS and then immediately placed in lysis buffer( Combine 0.2% (vol/vol) Triton X-100 and 2 U µl–1 RNase inhibitor).The lysis solution also contains free dNTPs and tailed oligo-dT oligonucleotides (30-nt poly-dT stretch and a 25-nt universal 5′ anchor sequence) that are prepared of the Reverse Trianscription reaction on polyadenylated RNA sequences. After the first reaction of reverse transcription, we used template-switching oligos to obtain the yield of full-length cDNA. Then, we performed RT-PCR to  maximize cDNA (After 90 min at 42 °C (the optimal conditions for Superscript II, according to the manufacturer), we raised the temperature to 50 °C for 2 min to promote the unfolding of any RNA secondary structures. We then lowered it to 42 °C again for 2 min and repeated this cycle a total of ten).After the first-strand reaction,We used tagmentation to quickly and efficiently construct sequencing libraries from the amplified cDNA. The fragments in the tagmented DNA library have an average size that ranges from 200 to 300 bp and then purified for Illimina. The  quantity of the libraries was assessed and defined by qPCR, respectively. All samples showed a concentration of higher than 3nM and were sequenced using HiSeq XTen (Illumina) with paired-end 150-bp sequencing.