Lightcycler 480

Amino acid starvation was carried out as described for ribosome profiling libraries, except cells were mock-treated with vehicle (ethanol or DMSO) in the no-CHX samples. Cells were harvested prior to adding CHX, and then 3, 6 and 9 min post-CHX by centrifuging 1 ml for 20 s at 11 000 × g. After aspirating the supernatant, cell pellets were immediately frozen in ℓN₂, and RNA was purified by phenol-chloroform extraction and ethanol precipitation (17 (link)). Residual genomic DNA was degraded using a TURBO DNA-free kit (Ambion). Oligo(dT) primers were annealed to mRNA, and cDNA was generated using AMV reverse transcriptase (Promega). NOP2 and ACT1 (nucleolar protein 2, ribi gene; actin, housekeeping reference) cDNAs were quantified on a Roche LightCycler 480 instrument using GoTaq (Promega) PCR reactions containing SYBR Green (Life Technologies), and relative abundances were calculated using the 2^−ΔΔCt method (18 (link)). The starvation plus rapamycin experiment was conducted as described above, except cells were incubated for 20 min in starvation medium containing 200 nM rapamycin (Sigma) or vehicle (DMSO) prior to adding CHX. All qPCR experiments were conducted with technical triplicates.

Total RNA was isolated by using a Reliaprep RNA Cell miniprep system (Promega). RNA (250–1000 ng) was reverse transcribed by using an Omniscript RT kit (Qiagen). Gene expression was analyzed by quantitative RT-PCR with a Roche Diagnostics LightCycler 480 in a 12-μL final volume with specific primers (10 μM) (Additional file 3: Table S2) and GoTaq PCR Master Mix (Promega). PCR amplification included a denaturation step (5 min at 95 °C) followed by 40 cycles of 10 s at 95 °C, 15 s at 60 °C, and 10 s at 72 °C.
For each PCR, duplicates for each cDNA were run in parallel, and serial dilutions of a cDNA mixture were tested for each primer pair to generate a standard linear curve, which was used to estimate the amplification efficiency. The relative mRNA expression for all genes analyzed was normalized to that of 18S RNA (used as the internal reference gene) and was analyzed by using the efficiency method with Light Cycler 480 software.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized using the GoScript Reverse Transcription (RT) system (Promega, Madison, WI, USA). RNA levels were measured by qRT-PCR on Roche Light Cycler 480 using the GoTaq qPCR Master Mix system (Promega, Madison, WI, USA). The expression of lncRNA and mRNA was normalized with GAPDH. The primer sequences are listed in the supplementary material.