Dntps n0447s

Cell pellets were collected and RNA was extracted using the Qiagen RNeasy kit following the manufacturer’s instructions. The reverse transcriptase reaction was performed with 1 µg RNA with random primers (C1181, Promega), dNTPs (N0447S, New England BioLabs), RNAse inhibitor (M0314L, New England Biolabs, and M-MuLV reverse transcriptase (M0253L, New England Biolabs). Quantitative PCR with reverse transcription was performed using Power SYBR Green PCR Master Mix (4368708, Thermo Fisher Scientific) on a Step One Plus Real-Time PCR machine (Applied Biosystems). The following program was used: 10 min at 95 °C followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Single melt curves were observed for all primers used in this study. Oligonucleotides used in this study are provided in Supplementary Table 4.

RNA was extracted using TRIzol reagent (15596010, Thermo Fisher Scientific) following the manufacturer’s instructions. The reverse transcriptase reaction was performed with 1 μg of RNA in the presence of random primers (C1181, Promega), dNTPs (N0447S, New England BioLabs), RNAse inhibitor (M0314L, New England Biolabs) and M-MuLV reverse transcriptase (M0253L, New England BioLabs). RT-PCR reactions were carried out on a Step One Plus Real-Time PCR machine (Applied Biosystems) using Power SYBR Green PCR Master Mix (4368708, Thermo Fisher Scientific). A list of all the primers used is provided in Supplementary Table 3. The following program was used: 10 min 95 °C denaturation and 40 cycles of 15 s 95 °C and 1 min 60 °C.