Miseq 2000 platform

DNA extraction for all assessed compartments was performed using the DNeasy PowerSoil kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol. The concentration of extracted DNA was determined with the Qubit HS assay (Invitrogen, Carlsbad, CA). Amplification of the SSU rRNA gene V4 region was performed using the primers 515F (5′-GTGCCAGCMGCCGCGGTAA′) and 806R (5′-GGACTACHVGGGTWTCTAAT′) [10 (link)]. Reactions were performed in triplicate of 5-ng DNA template in a solution containing DreamTaq buffer, 0.2 mM dNTPs, 0.5 μM of each primer, 0.75 μM of each mitochondrial (mPNA) and plastid (pPNA) peptide nucleic acid (PNA) clamps, and 1.25 U DreamTaq DNA polymerase as previously described ([45 ], further details in Table S4). PNA clamps have been shown to reduce plant plastid and mitochondrial DNA amplification in PCR reactions [58 (link)]. Triplicate PCR products were pooled together, barcoded with 10-base unique barcodes (Fluidigm Corporation, San Francisco, CA), and sequenced on an Illumina MiSeq2000 platform using a 500-cycle v2 sequencing kit (250 paired-end reads) at the Research Resources Center in the University of Illinois at Chicago. The raw SSU rRNA gene amplicon sequences have been deposited in the BioProject database (http://ncbi.nlm.nih.gov/bioproject) under accessions PRJNA666636.

16S rRNA gene sequences were amplified from each of the extracted DNA samples using a set of primers targeting the hyper-variable V3-V4 region (338F/806R) of the gene: 338F, 5′-barcode-ACTCCTACGGGAGGCAGCA-3′ and 806R, 5′-GGACTACHVGGGTWTCTAAT-3′. PCR amplification was performed as described in our previous study (Lu et al., 2016 (link)). DNA libraries were constructed using kits provided by Illumina Inc. according to the manufacturer’s instructions, and DNA sequencing was performed using the Illumina MiSeq 2000 platform (San Diego, CA, United States) at the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Zhejiang University, Hangzhou, China) according to standard protocols.

Swabs were transferred to the University of New South Wales (UNSW) in sterile cryo-tubes within liquid nitrogen. Microbial DNA was extracted from each swab using a 96-well PowerSoil DNA Isolation Kit (MO Bio Inc., Carlsbad, CA, USA) following the manufacturer's protocol. DNA extracts were qualified and quantified by agarose gel electrophoresis and spectrophotometry (NanoDrop 1000) and stored at −20°C. Samples were processed and sequenced using standard aseptic procedures and in a random order to avoid contamination and introducing any bias due to order of processing.
The extracted microbial DNA samples were amplified with PCR using the 16S rDNA primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCCGCGGCKGCTG-3′), containing V1 to V3 regions of the bacterial and archaeal 16S rRNA gene. PCR controls (no loaded sample) did not amplify DNA, suggesting no contamination during processing and amplification. Amplicons were purified (Zymo DNA-5 Clean Concentrator) before sequencing via the Illumina MiSeq 2000 platform at the Ramaciotti Centre for Genomics (UNSW).

To determine treatment effects on soil microbial community structure, microbial DNA was extracted from 0.25 g of soil (±0.5 g) using a MO BIO PowerSoil DNA Isolation Kit (MO BIO Laboratories Inc., Carlsbad, CA, USA) following the manufacturer's instructions. Extracted DNA was quantified using a Quant-iT PicoGreen ds DNA Assay Kit (Life Technologies, Carlsbad, CA, USA) and diluted with sterile water to 1 ng uL⁻¹ DNA. DNA extracts (10 μL per sample) were shipped overnight on dry ice to Argonne National Laboratory (Lemont, Illinois, USA) for amplicon library preparation of the V4 region of the 16S rRNA gene (515f and 806r primers; Caporaso et al., 2011 (link), 2012 (link)), following the Earth Microbiome Project protocol (Gilbert et al., 2010 (link)). Amplicons were subsequently sequenced using the Illumina MiSeq2000 platform.

The protocols for DNA extraction, V3–V4 amplification and sequencing were as described in our previous study [12]. Briefly, microbial DNA extracted using the Qiagen Mini Kit (Qiagen, Hilden, Germany), was quantified using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA), and diluted to 10 ng/μL for PCR amplification in a thermocycler (Eppendorf Mastercycler). DNA libraries were constructed according to the manufacturer’s instructions, and DNA sequencing was performed on the Illumina MiSeq 2000 platform (Shallowater, USA) at the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Zhejiang University, Hangzhou, China) according to standard protocols. The raw reads were deposited into the European Nucleotide Archive database (study accession no. PRJEB 27531).

Embryo hearts were dissected from euthanized E14.5 animals, and the ventricular tissue was isolated from the atrial chambers. RNA extracted from the ventricles using the RNeasy kit was subjected to the RNA-Seq pipeline at the Center for Cancer Computational Biology (Dana-Farber Cancer Institute). RNA was analyzed for quality control using Qubit fluorometric quantitation (Life Technologies) and Bioanalyzer (Agilent Technologies). RNA (50–100 ng) was converted into DNA libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs). The quality of the DNA libraries was assessed using the Qubit High Sensitivity DNA Kit (Life Technologies) and library size was determined using the Bioanalyzer High Sensitivity Chip Kit (Agilent Technologies). Libraries that passed quality control were diluted to 2 nM using sterile water and then sequenced on the MiSeq2000 platform (Illumina) at the concentration of 12 pM on a single read flowcell with 50 sequencing cycles.

For characterisation of microbial communities in all experiments, microbial DNA was extracted from each swab sample in a randomised order using a PowerSoil DNA Isolation kit (Qiagen) following the manufacturers protocol. DNA extracts were quantified using spectrophotometry (Nanodrop 1000) and stored at -20 °C until sequencing.
The extracted DNA samples were amplified with Polymerase Chain Reaction (PCR) using the 16 S rRNA gene primers 341 (F) (5’- CCTACGGGNGGCWGCAG-3’) and 805(R) – (5’-GACTACHVGGGTATCTAATCC-‘3), covering the V3-V4 regions of the bacterial and archaeal 16 S rRNA gene^{100 (link)}. The PCR conditions involved a pre-heating step to 95 °C for 3 min followed by 35 cycles of 95 °C for 15 s, 55 °C for 1 min and 73 °C for 30 s. Both positive (with known DNA sequence) and negative controls (nuclease-free water, control swabs) were used. The negative controls did not amplify DNA, suggesting no contamination on swabs/materials or during extraction and amplification. Agarose gel electrophoresis and Nanodrop 1000 were used to ensure the quantity and quality of the amplicons before they were sent for sequencing via the Illumina MiSeq 2000 platform at the Ramaciotti Centre for Genomics (UNSW, Sydney).