Custom primers

Total RNA was isolated (Fisher Scientific) from cultured cells after their respective treatments with ethanol or acetaldehyde. cDNA was synthesized using the QuantiMiR^TM RT kit (System Biosciences, Mountain View, CA) as per the manufacturer’s instructions. Real-time PCR reaction mixes were created using FastStart Universal SYBR Green Master Mix (Roche Diagnostics), and run on a StepOnePlus^TM Real-Time PCR System (Applied Biosystems) using the following program: 50 °C for 2 min, 95 °C for 10 min, 95 °C for 30 s, and 60 °C for 1 min, for 40 cycles. Experiments were analyzed using the ddCt method. U6 primers and a Universal Reverse Primer were used from the QuantiMiR^TM RT kit, and custom primers (Eurofins MWG Operon) were ordered using the following sequences: miR-3178: 5’-GGGGCGCGGCCGGATCG-3’, miR-675: 5’-TGGTGCGGAGAGGGCCCACAGTG-3’, miR-934: 5’-TGTCTACTACTGGAGACACTGG-3’, miR-101: 5’-CAGTTATCACAGTGCTGATGCT-3’, miR-1266: 5’-CCTCAGGGCTGTAGAACAGGGCT-3’, miR-30a: 5’-TGTAAACATCCTCGACTGGAAG-3’, miR-3164: 5’-TGTGACTTTAAGGGAAATGGCGAA-3’, and miR-3690: 5’-ACCTGGACCCAGCGTAGACAAAG-3’.

Each SISO system is comprised
of (1) a single transcription factor expressed on the pLacI plasmid
(Novagen), which contains the p15a origin (copy number 20–30/cell),
and (2) a super folder green fluorescent protein (GFP) reporter expressed
on the pZS*22-sfGFP plasmid which contains the pSC101 origin (copy
number 3–5/cell). Chloramphenicol and kanamycin resistance
genes were used as selection markers for transcription factor and
reporter plasmids, respectively. The transcription factor and reporter
plasmids were obtained from previous works (Rondon et al., Groseclose
et al.) and when necessary, ADR or operator variants were cloned using
site-directed mutagenesis PCR (Phusion DNA Polymerase, NEB) with custom
primers (Eurofins Genomics), followed by kinase, ligase, and DpnI reactions (KLD enzyme mix, NEB). The reactions were
transformed into chemically competent DH5α cells (huA2 Δ(argF-lacZ)U169
phoA glnV44 φ80Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1
hsdR17; New England Biolabs) and plated on LB agar with an appropriate
antibiotic. A transformant was cultured overnight in LB broth (Fisher
BioReagents) and mini-prepped (Omega Bio-Tek) to yield each plasmid,
and the sequence was confirmed with DNA sequencing (Eurofins Genomics).