Advantage hf 2 dna polymerase

After rRNA subtraction and purification (described above), primers with Tm higher than 60 °C were used directly to reverse transcribe the extracted DMS-modified RNA as described above without fragmentation and linker ligation. For the whole genome rt-PCR library, primers were designed to cover the entire 30 kb with ~50 nt overlap (Supplementary Data 5). The cDNA was then purified with Oligo Clean and Concentrator −5 (Zymo) following the manufacturer’s instructions. Two microlitres of cDNA were amplified using Advantage HF 2 DNA polymerase (Takara) for 30 cycles according to the manufacturer’s instructions and purified by DNA Clean and Concentrator −5 (Zymo) following the manufacturer’s instructions. RNA-seq library for 150 bp insert size was constructed following the manufacturer’s instruction (NEBNext Ultra^™ II DNA Library Prep Kit) and sequenced on a Nextseq system (paired-end run, 150 cycles).

To reverse transcribe, rRNA-depleted total RNA or in vitro-transcribed RNA purified from the previous steps was added to 4 µl 5× FS buffer, 1 µl dNTP, 1 µl of 0.1 M DTT, 1 µl RNase Out, 1 µl of 10 µM reverse primer (5′-TCGTTGAAACCAGGGACAAG-3′, purchased from IDT) and 1 µl TGIRT-III (Ingex). The reaction was incubated for 1.5 h at 60 °C. Then, to degrade the RNA, 1 µl of 4 M NaOH was added and incubated for 3 min at 95 °C. The cDNA was purified in 10 µl water using the Oligo Clean and Concentrator^TM kit (Zymo). Next, 1 µl of cDNA was amplified using Advantage HF 2 DNA polymerase (Takara) for 25–30 cycles according to the manufacturer’s instructions with 5′-leader primer set listed in Supplementary Table 3. The PCR product was purified using E-Gel^TM SizeSelect^TM II 2% agarose gel (Invitrogen). RNA-seq library for 300 bp insert size was constructed following the manufacturer’s instructions (NEBNext Ultra^TM II DNA Library Prep Kit). The library was loaded on iSeq-100 Sequencing flow cell with iSeq-100 High-throughput sequencing kit and library was run on iSeq-100 (paired-end run, 151 × 151 cycles).