Rnase free dnase

Birds were culled by cervical dislocation, caecal pouches were removed immediately post-mortem using sterile scissors and the caecal contents recovered by squeezing into sterile cryovials (one per chicken) containing Bacterial Protect RNA reagent (Qiagen, Germany) at an approximate ratio of 1:1. Each sample was stored and transported to the laboratory in a portable freezer at −20°C and then stored at −80°C prior to further processing. Total genomic DNA (gDNA) was extracted using a QIAamp Fast DNA Stool Mini kit (Qiagen, Germany) following the manufacturer's instructions with minor modifications. Briefly, 500 μL of caecal content mixed with Bacterial Protect RNA reagent was added to 1 mL InhibitEX buffer and homogenized by vortexing at maximum speed (3,000 rpm) for 5 min, then incubated at 80°C for 10 min. The mixture was centrifuged at 2,600 g for 1 min to remove residual solid material and 600 μL of supernatant was processed as recommended by the manufacturer. gDNA was treated with DNase free RNase (Macherey-Nagel, Germany) to remove contaminating RNA. gDNA concentration and quality were assessed using a Qubit 2.0 fluorometer (Invitrogen, ThermoFisher scientific, MA) and 0.8% (w/v) agarose (SeaKem® LE Agarose, Lonza, Switzerland) gel electrophoresis (in 0.5x TBE buffer) respectively. gDNA was stored at −20°C until further processing.

The luminal content collected from each section of the intestinal tract was thawed on ice and ~300 µL used for metagenomic DNA extraction using the QIAamp Fast DNA Stool Mini Kit (QIAGEN, Germany) as per the manufacturer’s instructions. Further, metagenomic DNA was treated with DNase-free RNase (Macherey-Nagel, Germany) to remove any contaminating RNA. The quality and quantity of metagenomic DNA was assessed using agarose gel electrophoresis and a Qubit 3.0 Fluorometer (Invitrogen, Life Technologies, USA), respectively. Metagenomics DNA was stored at −20 °C until further processing. The hypervariable region V3–V4 of the 16S rDNA gene was used to assess microbial diversity between the high and low FCR groups. Amplified amplicons and shotgun metagenomics libraries were made using a Nextera XT DNA library preparation kit (Illumina, USA) according to the manufacturer’s instructions. These libraries were sequenced on an Illumina MiSeq platform using a 2 × 250 sequencing kit.

At each location, five apparently healthy chickens were selected at random from each group, caught and euthanized by cervical dislocation at 42 days of age. Both caecal pouches were opened immediately using sterile scissors and the contents were recovered into sterile cryovials containing Bacterial Protect RNA reagent (QIAGEN, Germany) at an approximate 1:1 ratio (w/v). Each sample was immediately stored in a portable freezer at − 20 °C, transported to the laboratory and stored at − 80 °C.
Total genomic DNA was extracted from the pooled caecal contents of each individual chicken using the commercially available QIAamp Fast DNA Stool Mini kit (QIAGEN, Germany) following the manufacturer’s instructions with some modifications. Briefly, 300 μL of caecal content with Bacterial Protect RNA reagent was added to 1 mL of InhibitEX buffer, vortexed at 2800 rpm for 1 min to homogenise and incubated at 80 °C for 10 min. The mixture was then centrifuged at 2600 g for 30 s to remove residual solid material and 600 μL of supernatant was processed as recommended by the manufacturer. DNA was treated with DNase free RNase (Macherey-Nagel, Germany) to remove contaminating RNA. DNA concentration and quality were assessed using a Qubit 2.0 fluorometer (Invitrogen, ThermoFisher scientific, MA) and gel electrophoresis. DNA was stored at − 20 °C until further processing.