Nanodrop quantification

Mixed-staged animals with 4-h extract treatment or without were collected in TRIzol (Invitrogen) and RNA was extracted using isopropanol/ethanol precipitation. The quantity and quality of RNA were estimated by NanoDrop quantification (Thermo Scientific) and gel electrophoresis, respectively. cDNA was synthesized using Superscript IV (Invitrogen) with Oligo(dT) primers using 2 to 3 µg RNA as per the manufacturer’s instructions. Real-time PCR was performed using qPCR primer pairs listed in SI Appendix, Table S2 and LightCycler480 SYBR Green I Master (Roche) in a LightCycler480 instrument. Ct values were derived using the LightCycler480 software and second derivative maximum method. All experiments were performed in biological triplicates. The expression levels of old-1, flor-1, and chil-27 were normalized with the values obtained for the reference gene pmp-3. The efficiency of each set of primers and calculation of levels of induction was calculated by the 2^−ΔΔCt method.

After co-incubation with either LYBHI or F. prausnitzii SN, HT-29 cells were collected and RNA-purified for DNA transcriptomic hybridization. Total RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, USA) and purified by on-column digestion of DNA with DNase I as recommended by the manufacturer to eliminate residual genomic DNA. RNA concentration was determined by Nanodrop quantification (Thermo Fisher Scientific Inc., France). RNA quality was checked on an Agilent 2100 Expert Bioanalyzer (Agilent Technologies, France). Only RNAs with a RIN > 8 were used for transcriptomic and qRT-PCR experiments.

C. elegans at L4 stage were treated with extract for 4 h and total RNA was extracted from animals grown at the appropriate stage/exposure time using the TRIzol reagent (Invitrogen) and isopropanol/ethanol precipitation. NanoDrop quantification (Thermo Scientific) and gel electrophoresis was used to assess the quantity and quality of RNA. 2 μg of RNA from all samples was subjected to DNase (Promega) treatment and cDNA was synthesized using Superscript IV (Invitrogen) with Oligo(dT) primers as per manufacturer’s instructions. Real-time PCR was performed using LightCycler480 SYBR Green I Master (Roche) in a LightCycler480 instrument and Ct values were derived using the LightCycler480 software and second derivative maximum method. Expression levels of chil-27 were normalized with the values obtained for the reference gene pmp-3. The efficiency of each set of primers and calculation of levels of induction was determined as described previously (Pfaffl, 2001 (link)). Experiments were performed in biological triplicates and oligos used are listed in the Key Resources Table.