For each biological replicate, four 60 mm NGM plates of mixed-stage N2 worms were washed off of plates with M9 buffer into a single 15 mL conical tube. Worm suspensions were spun-down at 1300xg for 5 min, supernatants were aspirated, and worm pellets were transferred to 2 ml RNase-free micro-centrifuge tube. Worm pellets were washed with 1 ml of M9 buffer two additional times to remove residual bacteria. Worm pellets (~ 100 ul) were snap frozen in liquid nitrogen. RNA was extracted using TRIzol™ (Thermo Scientific #15596026) as per manufacturer’s directions.
cDNA was synthesized using Maxima H Minus First Strand cDNA Synthesis Kit (Thermo Scientific #K1681) following manufacturer’s directions. Briefly, ~ 500 ng of RNA from each biological replicate was combined with 1 ul of dsDNase, 1 ul of 10x dsDNase buffer and brought-up to 10 ul with nuclease-free H2O. This mixture was incubated at 37 °C for 2 min and then immediately returned to ice. Then, to the RNA containing tube, 1 ul random hexamer primers, 1 ul of 10 mM dNTP, 4 ul 5x RTase buffer, 1 ul of Maxima H Minus reverse transcriptase, and 3 ul of nuclease-free H2O were added. The cDNA synthesis reaction was run in a thermocycler: 25 °C for 10 min, 55 °C for 30 min, 85 °C for 2 min, 4 °C hold. Following cDNA synthesis, cDNA was diluted to 50 ul with nuclease-free H2O and stored at -80 °C.
Droplet digital PCR (ddPCR)
cDNA dilution: cDNA was diluted in nuclease-free water to run in the dynamic range of ddPCR. Initially, all targets were diluted 1:200. This dilution fell within the dynamic range for the protein-genes. However, at this dilution the rRNA levels saturated the capacity of ddPCR. From this initial result it was determined that a 100x further dilution would be sufficient to accurately quantify rRNA levels. Consequently, cDNA was diluted 1:20,000 for reactions that measured rRNA targets.
ddPCR reaction: In the wells of a Eppendorf™ 96-Well twin.tec™ PCR Plate (Fisher Scientific # E951020303), 2 ul of diluted cDNA was combined with 10 ul of nuclease-free water, 12.5 ul of QX200™ ddPCR™ EvaGreen Supermix (Bio-Rad #1864034), and 0.25 ul of each corresponding forward and reverse primer (10uM working stock). Droplets were generated on a QXDx Automated Droplet Generator. Following droplet generation cDNA was amplified: 95 °C for 5 min, 39 cycles of 95 °C for 30sec then 58 °C for 1 min, 4 °C for 5 min, 90 °C for 5 min, 10 °C hold. Following cDNA amplification, droplets were read on a QXDx Droplet Reader.
12S rRNA: CTTGTTCCAGAATAATCGGCTAGACTTG / CTAACCAGGTACTAATCTGCTTTGTTCAAC
16S rRNA: CAGTCTTAGCGTGAGGACATTAAGGTA / CTAACCAATAACTTCATTCATACTGGAACTC
COI: CAGCAGGGTTAAGATCTATCTTAGGTGG / CGATCAGTTAACAACATAGTAATAGCCCC
COII: CTAGATCAATTAAGTTTAGGTGAACCACG / CCAAGCATGAATAACATCAGCAGATG
COIII: GCTTGAGGTAAGGATATTGCTATAGAAGG / GTGTACTGGTACTAGAGCAGCATC
ND1: GCCATCCGTGCTAGAAGACAAAG / CCTCTAACTAACTCCCTTTCACCTTCAG
ND4: ATTTCCAATTTATTTTTTACATCTTTGATTACC / CCCGCTGTGCCTAATTTTAATAG
ND5: GATCTTGGTTACCCAAAGCTATAAGAGC / GTGTCCTCAAGGCTACCACCTTCTTC