GSM8045952: FGO_STARR_seq_rep2; Mus musculus; OTHER
8-week-old female mice were intraperitoneally injected with PMSG. Oocytes were harvested in M2 medium 48 h after PMSG. The STARR-seq plasmid library was purified and injected into the pronucleus of FGOs cultured with milrinone. After 24 hrs, the FGOs were lysed in hypotonic lysis buffer (Vazyme, N712), and the polyadenylated mRNAs were captured by the Oligo (dT) primers. After 3 min at 72 °C, the Smart-seq2 reverse transcription reactions were performed. After pre-amplification and AMPure XP beads purification, cDNAs were amplified with junction PCR primers (forward: 5`-TCGTGAGGCACTGGGCAG*G*T*G*T*C, reverse: 5`-CTTATCATGTCTGCTCGA*A*G*C, *:phosphorothioate bond), which specifically enriches reporter transcripts and excludes STARR-seq plasmids, as the junction forward primer only binds to the spliced intron of the reporter transcripts. Then the PCR products were cleaned up with AMPure XP beads, and we performed sequencing-ready PCR to add Illumina i5 and i7 indexes for deep sequencing. All libraries were sequenced on Illumina HiSeq 2500 according to the manufacturer's instruction.