Smart-seq2 of Sus scrofa: 4-cell embryos
RNA-seq libraries of porcine 4-cell embryos and FACS-sorted cells were prepared using a modified Smart-seq2 protocol (Picelli et al., 2014). Either 10 zona-free embryos or 500-1000 sorted cells were transferred into 4 ?L of lysis buffer containing 0.1% Triton X-100 (Sigma, 93443), 5  10?? M oligo-dT primer (5-AAGCAGTGGTATCAACGCAGAGTACT??VN-3), 1  10?? M dNTPs (NEB, N0447S), and 4 U RNase inhibitor (Takara, 2313A). The samples were incubated at 72C for 3 minutes for RNA denaturation. Subsequently, 5.7 ?L of reverse transcription mixture was added, comprising 100 U SuperScript II reverse transcriptase (Invitrogen, 18064014), 1 Superscript II first-strand buffer, 5  10?? M DTT, 1 M betaine (Sigma, 61962), 6  10?? M MgCl? (Sigma, M1028), 1  10?? M TSO (5-AAGCAGTGGTATCAACGCAGAGT-3), and 10 U RNase inhibitor. Reverse transcription was carried out at 42C for 90 minutes to synthesize cDNA. The resulting cDNA was pre-amplified using KAPA HiFi HotStart ReadyMix (Roche, KK2601) and IS PCR primers (5-AAGCAGTGGTATCAACGCAGAGT-3) for 16-18 cycles. The amplified cDNA was purified using VAHTS DNA Clean Beads (Vazyme, N411-01). Finally, cDNA libraries were constructed using the TruePrep DNA Library Prep Kit (Vazyme, TD502) following the manufacturer's protocol.