5300 fragment analyzer system

Total RNA was isolated using the RNeasy Midi Kit (QIAGEN). RNA-seq quality and quantity controls were performed using a Nanodrop spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific) and a 5300 Fragment Analyzer system (Agilent). Library preparation and sequencing was carried out by the GeT core facility (Toulouse, France; http://get.genotoul.fr) with the TruSeq Stranded total RNA Kit (Illumina) according to the manufacturer's instructions. Sequencing was performed using a HiSeq 3000-HWI-J00115 according to the manufacturer's protocol.

After the worms were exposed to P. aeruginosa for the experimentally designated times (ranging from 0 to 24 h), they were collected in M9 buffer and decanted 3 times in 15-mL tubes to allow digestion of bacteria inside the worms and to separate adults from any larval progeny (no 5-Fluoro-2′-deoxyuridine was utilized in the P. aeruginosa assays). Following washing, the worm pellets were treated with guanidinium thiocyanate and RNA isolated using phenol/chloroform and ethanol precipitation. The obtained RNA was quantified and subjected to capillary electrophoresis using a 5300 Fragment Analyzer system (Agilent, California, United States of America) using a “standard sensitivity” RNA assay. The obtained electropherograms were analyzed using the ProSize 2.0 software (Agilent) to quantify the relative concentration of distinct rRNA species.
For the analysis of the total RNA content per worm (S1B Fig), sets of 20 adult worms were exposed to the designed treatment for 24 h. Following exposure, the worms were washed with M9 buffer, and individual worms were manually transferred into 0.25-mL guanidinium thiocyanate containing a fixed amount of a 500-nt spike-in RNA (in vitro translated). RNA was isolated by phenol–chloroform extraction followed by ethanol precipitation. RNA was profiled with capillary electrophoresis as described above.

In general, one isolate per animal was included and isolates from the same animal were included only if they produced differential randomly amplified polymorphic DNA (RAPD) typing banding patterns (Power, 1996 (link)). Primers and PCR conditions were as described previously (Baker et al., 2010 (link)) with the only difference being that amplicon fragments were separated on a 5300 Fragment Analyzer System (Agilent, Santa Clara, CA, United States). Nineteen C. perfringens isolates were from broiler chicken intestinal samples collected during NE outbreaks, however, the presence of NE lesions was not recorded for these intestinal tracts, while three isolates were from healthy broiler chicken intestinal samples. Twenty-one isolates were from dairy cow intestinal samples with HBS, and one isolate was from a fecal sample of a dairy cow with HBS.

Biopsies from the descendent colon were obtained during routine colonoscopy screening for cancer from patients negative from neoplasms. The biopsies consisted of mucosal samples, which includes epithelium, lamina propria and basal membrane. Samples were maintained in RNAlater (Thermo Fisher Scientific, Waltham, MA, USA), to preserve RNA integrity. Then, samples were immediately transported to the laboratory. The handling of tissue was carried out under strictly aseptic conditions and stored at −80 °C. RNA purification was performed using RNeasy-Tissue Mini-Kit (Qiagen, Hilden, Germany). Total RNA was quantified by Qubit^® RNA BR Assay kit (Thermo Fisher Scientific) and the integrity was checked by using the RNA Kit (15NT) on 5300 Fragment Analyzer System (Agilent, Santa Clara, CA, USA). Colon transcriptomics was performed with Affymetrix HUGENE 2.0 ST ARRAY FORMAT 100.