Universal sybr green fast qpcr

Yeast strains were grown to an OD of approximately 0.6 to 1 for collection. Three biological replicates were used for each set of experiments (except 2 replicates for RNH1 levels, S1B Fig). Cells were treated with 50 to 100 U of zymolyase in a 1 M sorbitol/100 mM EDTA solution. Cells were lysed and RNA was extracted using the RNeasy kit and RNase-free DNase set (QIAGEN). cDNA was prepared using the Superscript First Strand Synthesis kit (Life Technologies); oligo d(T) primers or locus specific primers (see Table P in S1 Tables) were used for priming during reverse transcription. RT-PCR samples were analyzed using qPCR (performed in technical duplicate with the average shown) with SYBR Premix Ex Taq II (Tli RNase H Plus) (Takara Bio), Power SYBR Green PCR Master Mix (Applied Biosystems), or 2X Universal SYBR Green Fast qPCR (ABclonal) on a QuantStudio 6 real-time PCR system (Applied Biosystems).

RNA was isolated from HEK 293 cells using a Quick-RNA MiniPrep Kit (Zymo Research, R1055, Irvine, CA, USA). cDNA was synthesized using LunaScript RT SuperMix (NEB, E3010, Ipswich, MA, USA), followed by qPCR using 2x Universal SYBR Green Fast qPCR (Abclonal, RK21203, Woburn, MA, USA). The following primers were used: SETD2-forward CTCCTCCCAAACCAAAAACC, SETD2-reverse GAGTTCCCAGGTCCATCTCA; LC3B-forward GAGAAGCAGCTTCCTGTTCTGG, LC3B-reverse GTGTCCGTTCACCAACAGGAAG; FIP200-forward CAGCACAAAGTTTGGATGAAATGTC, FIP200-reverse CCTGCTGTACTTTCCATCCTTGG; ULK1-forward GCAAGGACTCTTCCTGTGACAC, ULK1-reverse CCACTGCACATCAGGCTGTCTG; ATG13-forward CAGAACTGCTGGTGAGGACACT, ATG13-reverse AGCAGGCTGATAGGAAAGGCGA; GAPDH-forward GAGTCAACGGATTTGGTCGT, GAPDH-reverse TTGATTTTGGAGGGATCTCG.