Mobio powersoil kit

Fecal samples were collected from pregnant dams on the day that ABX treatment ceased and were stored at −80°C prior to DNA extraction.
Fecal samples were homogenized and DNA was extracted using a MoBio Power Soil Kit (MoBio Laboratories, QIAGEN). Samples were stored at −80°C prior to further analysis as described below.

Rectal fecal samples from each pig were collected at the farm of origin (day-18 p.i.) and in our stables on days 0, 14, and 28 p.i. and immediately cooled down on ice; individual 0.25 g subsamples were stored at −80°C. At day 28 p.i., 0.25 g subsamples of thoroughly homogenized digesta from the ileum (taken 10 cm oral to the ileo–cecal junction), cecum (blind end), proximal (20 cm aboral from the ileo–cecal junction), and distal colon (midway between cecum and rectum) were collected from each pig and kept on ice before transfer to −80°C within 1 h. All samples collected during the study were subjected to DNA extraction using Mobio PowerSoil kit (Mobio Laboratories, CA, United States), following the manufacturer’s protocol. The extracted DNA was stored at −20°C until further analysis. Samples from the proximal colon were also investigated by gas chromatography for SCFA concentrations, as described by Myhill et al. (2018) (link).

DNA extraction for the microbiome analysis from the fecal samples was processed using previously published methods [47 (link)]. Briefly, lyophilized fecal samples were homogenized and 50 mg/sample was used for DNA extraction with the MoBio PowerSoil Kit (MoBio Laboratories Inc., Solana Beach, CA, USA). Extracted DNA samples were stored at −20 °C until concentration and quality checking on a NanoDrop 2000 (Thermo Fisher Scientific, USA). V4 hypervariable region amplification of the 16S rRNA gene and amplicon sequencing on an Illumina MiSeq platform (Illumina Inc., San Diego, CA, USA) observed guidelines by the Earth Microbiome Project [48 (link),49 (link)] and utilized the 515F/806R (Parada/Apprill) primer pair. Raw, single-end FASTQ forward sequence reads were analyzed using QIIME2 [47 (link)]. Feature tables of absolute relative abundance amplicon sequence variants (ASV) were constructed for each sample using reads from the DADA2 pipeline [50 (link)]. Taxonomic identities for ASV representative sequences were assigned with Naive Bayes classifiers independently trained on 99% OTU reference collections bound by the 515F/806R (Parada/Apprill) primer pair and trimmed to 248bp extracted from either the Greengenes 13_8 [51 (link)] or SILVA 132 [52 (link)] marker gene databases. Data were exported from QIIME2 to R for further analysis [53 ].

Extractions were carried out using sterile technique in a laminar flow hood. For pooled tissue extractions, equal sections of leaf tissue (50 mm²) and root tissue (12.5 mm³ plus 10 mm of lateral roots) were collected from each individual sample per location and pooled prior to extraction. Control (blank) samples were collected for each batch of extractions by swabbing tools and surfaces and then extracting DNA from the swab head.
All DNA samples were extracted using the Mo Bio PowerSoil kit (Mo Bio Laboratories, Inc.). Phyllosphere and ectorhizosphere DNA was extracted from up to 0.25 g of wash pellets according to the standard kit protocol. Leaf and root tissues were ground to powder or sawdust consistency in liquid nitrogen using sterile mortars and pestles. Leaf and root DNA was extracted from 20 mg (leaf) or 100 mg (root) of ground tissue with the following modification to the standard protocol: tissue was incubated at 65°C for 10 min in extraction buffer and then vortexed for 1 min, followed by a second 10-min incubation (as described under “alternative lysis methods” in the kit protocol). Control DNA was extracted by placing whole swab heads directly into extraction tubes. Extracted DNA was eluted in PCR-grade water and stored at −20°C pending library preparation.

Root and shoot samples were lyophilized for 48 hours using a Freezone 6 freeze dry system (Labconco, Fisher Scientific, Hampton, NH) and pulverized using a tissue homogenizer (MP Biomedicals, Solon, OH). Agar from each plate was stored in a 30 ml syringe (Fisher Scientific, Hampton, NH) with a square of sterilized Miracloth (Millipore) at the bottom and kept at −20°C for a week. Syringes were then thawed at room temperature, and samples were squeezed gently into 50 ml tubes. Samples were centrifuged at maximum speed for 20 minutes, and most of the supernatant was discarded. The remaining 1 to 2 ml of supernatant containing the pellet was transferred into clean microfuge tubes. Samples were centrifuged again, supernatant was removed, and pellets were stored at −80°C until DNA extraction.
DNA extractions were carried out on ground root and shoot tissue and agar pellets using 96-well-format MoBio PowerSoil Kit (MOBIO Laboratories; Qiagen, Hilden, Germany) following the manufacturer’s instruction. Sample position in the DNA extraction plates was randomized, and this randomized distribution was maintained throughout library preparation and sequencing.

Total genomic DNA was extracted from 0.5 g of soil samples using the MoBio Powersoil Kit (MoBio Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The DNA quantity and quality of each sample was determined by using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) and electrophoresis (1.0% agarose gel, including a 1 kb plus ladder). The DNA samples were stored at − 80℃ until use. Metagenomic library preparation and sequencing were performed following the manufacturer’s protocol at Biozeron, Shanghai, China. Fifteen rhizosphere soil DNA were selected for metagenomic sequencing to evaluate the microbial community structure and function. Metagenomic libraries were constructed using a TruSeq™ DNA Sample Prep Kit (Illumina, USA) according to the manufacturer’s protocol. The metagenomic DNA was sonicated to the 450 bp size range using a Covaris M220. The metagenomic libraries were sequenced on a HiSeq 2500 sequencer (Illumina, USA), and 150-bp paired-end reads were generated.