Luna universal master mix

Total RNA was extracted from cells using Monarch total RNA miniprep kit (T2010S; New England Biolabs, Ipswich, MA). cDNA was synthesized with the AffinityScript QPCR cDNA Synthesis Kit (Agilent Technologies, Santa Clara, CA). Quantitative real-time PCR (qPCR) was performed using Luna universal master mix (M30043; New England Biolabs) on Mx3005P QPCR system (Agilent Technologies). The PrimeTime Predesigned qPCR Assay probes used (Integrated DNA Technologies, Coralville, IA) and were LDLR: hs.pt.53.24904291, transferrin receptor protein: Hs.PT.58.22906586, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR): hs.pt.56.41105492, PCSK9: hs.pt.53a.19328431, fatty acid synthase: Hs.PT.58.20384174, SREBP-1c: Hs.PT.58.25035932, SREBP-2: Hs.PT.58.4533543, and ceraldehyde-3-phosphate dehydrogenase: Hs.PT.39a.22214836. 25 ng DNA template was used, and the samples were analyzed as duplicates in four separate experiments. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was used for normalizing the amount of target mRNA. Relative mRNA expression was calculated using the 2^−ΔΔCt method (28 ).

For each condition, one 100 mm dish was used for total RNA extraction. Cells were differentiated and stressed before isolating RNA using RNeasy Mini Kit (Qiagen, Cat #74104) with DNase digestion (Qiagen, cat #79254). CDNA synthesis was done using MiniAmp Thermal Cycler (Applied Biosystems). Purified RNA sample concentrations were measured by Nanodrop and 1 µg RNA was incubated with dNTP (New England Biolabs, cat #N0447S), random hexamers (Thermo Scientific, cat #SO142), and nuclease free water (Invitrogen, cat #AM9930), at 65 °C for 5 min. After the first incubation, 5 × First Strand Buffer, DTT (Invitrogen cat #18080093) and RNase inhibitor (Invitrogen, cat #AM2694) were added to the mixture and incubated at 25 °C for 2 min. After the second incubation, 1 µL Superscript III Reverse Transcriptase (Invitrogen, cat #18080093) was added and the mixture was incubated at 25 °C for 10 min, 42 °C for 50 min, and 70 °C for 15 min. CDNA was stored at − 20 °C overnight or used immediately at a dilution of 1:20 in nuclease free water for RT-PCR experiments with Luna Universal Master Mix (New England Biolabs, cat #M3003L), nuclease free water, and custom ATP5f1b and CHGb primers (ITD). For positive and negative controls, 18S ribosomal subunit and tau primers were used (see Supplemental).

Total RNA was isolated from ~500 day 1 adult hermaphrodites raised on E. coli OP50 bacteria maintained at indicated conditions. RNA was extracted and purified with a RNeasy Mini kit (QIAGEN), and subjected to an additional DNA digestion step. 1 μg DNase-treated total RNA was used as a template for cDNA synthesis using anchored-oligo (dt)₁₈ primer and M-MLV reverse transcriptase (Roche). qPCR was performed using Luna Universal master mix (New England Biolabs) in an LC480 LightCycler (Roche). A standard curve was obtained for each primer set by serially diluting a mixture of different complementary DNAs and the standard curves were used to convert the observed CT values to relative values. mRNA levels of target genes were normalized to the mean of the housekeeping gene act-4. Primers used for qPCR are listed in Supplementary Data 7.

cDNA from mice brain (used here for qRT-PCR analysis) was prepared from mice tissue’ samples which were previously collected^{96 (link),97 (link)}. Tissue RNA was extracted with TriReagent (Sigma, T9424) according to supplier instructions. RNA concentration and quality were determined using Nanodrop (PeqLab) and cDNA was synthesized with the iScript cDNA synthesis kit (Bio-Rad, 1708891) following manufacturer’s protocol. Each 9 µl reaction for q-RT-PCR was made of 4 µl diluted cDNA, 0.25 µl of each primer (from 25 µM stock) and 4.5 µl of Luna Universal Master Mix (New England Biolabs, M3003S). The q-RT-PCR reactions were run on the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) using the BioRad iQ5.2 Software. qPCR results were analyzed using the DDCt method relative to the mean of housekeeping genes (Hprt, Gapdh and ActB). Each biological data point represents the average of two technical triplicates. Primer pairs used are listed in Supplementary Table 6.

Total RNA was extracted using Aurum™ Total RNA Mini Kit (BioRad, #7326820, Hercules, CA, USA) according to the manufacturer’s instructions. RNA quantification was performed using a NanoDrop (NanoDrop Lite, Thermo Scientific, Waltham, MA, USA). Total RNA (40 ng) from each sample was used for cDNA synthesis using the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher, #4374966, Waltham, MA, USA) on a T100 thermal cycler (BioRad, Mississauga, ON, Canada). For RT-qPCR, reactions were prepared using 1 μL of cDNA template (22.5 ng/μL) and 500 nM of forward and reverse primers in a final volume of 10 μL. PCR amplification was performed using an QuantStudio 3 Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with Luna Universal Master Mix (New England Biolabs Ltd., #M3003, Whitby, ON, Canada). For normalization, the housekeeping gene, GAPDH, was used as a reference. The rat-specific primer sequences used are as follows: XBP1 forward, 5′-ACGAGAGAAAACTCATGG -3′ and reverse, 5′-ACAGGGTCCAACTTGTCC-3′, and GAPDH forward, 5′-CATCAACGACCCCTTCATTGACCTCAACTA-3′ (Invitrogen, Life Technologies, Eugene, OR, USA) and reverse, 5′-TCCACGATGCCAAAGTTGTCATGG -3′ (Invitrogen, Life Technologies, Eugene, OR, USA). The comparative threshold (CT) method was used to quantify gene expression.

Total RNA was isolated from cells with TRIZOL (Invitrogen) according to the manufacturers’ protocol. cDNAs were made using the QuantiTect kit (Qiagen). QPCR was performed using an ABI Step One-Plus System with LUNA Universal master mix (NEB).