Nanophotometer spectrophotometer

Total RNA quality was assessed using NanoPhotometer spectrophotometer, Qubit 2.0 Fluorometer (Life Technologies), and Agilent 2100 Bioanalyzer. High quality RNA was then subjected to library preparation using a NEBNext^® Ultra^TM Directional RNA Library Prep Kit for Illumina^® (New England BioLabs) according to the manufacturer’s instructions with input ≥1 μg of total RNA. Paired-end sequencing with a read length of 150 bp was performed on Illumina HiSeq 2500 platform, yielding at least 50 (71.35 ± 11.10, mean ± s.d.) million sequence reads mapped to 16,052 ± 996 RefSeq entries per sample. Image analysis, base calling, and quality check were performed with Illumina data analysis pipeline RTA v1.18.64 and Bcl2fastq v1.8.4. The sequence reads were provided in compressed Sanger FASTQ format.

DNA was extracted from sediment samples using MoBio PowerSoil^® DNA Isolation Kit according to the SDS method (Clegg & Griffiths, 1997). Each replicated sample (5 g) was extracted in triplicate, and the DNA samples were subsequently pooled. The concentrations of DNA were determined by using a NanoPhotometer spectrophotometer and a Qubit 2.0 Fluorometer (Life Technologies, CA). The different regions of the 16S rRNA gene were chosen and then amplified by the corresponding primers: 338F‐533R for the V3 regions, 341F‐805R for V3+V4 regions, and 967F‐1046R for the V6 regions. The V3‐V4 region of the 16S rRNA gene was amplified from the bacterial DNA by polymerase chain reaction (PCR) using the modified primers 341F (5′‐CCTACGGGNGGCWGCAG‐3′) and 805R (5′‐GACTACHVGGGTATCTAATCC‐3′) as described elsewhere (Vasileiadis et al., 2012), which was based on the design method described previously (Wang & Qian, 2009). The index sequences were added, and enrichment after the extraction was completed. The Qubit 2.0, Agilent 2100 and Bio‐Rad CFX 96 instruments were used to quantify the concentration and purity of the library samples to ensure their quality. After these steps were complete, the library was sequenced on an Illumina MiSeq platform by using the 250 paired‐end (PE) protocol.

Real-time PCR experiments were conducted as described (Xiao et al., 2019 (link)). Twenty-four hours after surgery, the mouse hearts were taken out and stored in liquid nitrogen, and then liquid nitrogen was added to pre-cool the mortar. One ml of lysate was added to each tissue sample, and the tissue was ground. The homogenate was separated and the supernatant was removed. RNA was isolated with chloroform, precipitated with isopropyl alcohol, washed with 75% ethanol and was dissolved with ultrapure water. The RNA concentration, purity and integrity were separately measured using Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, CA, United States), the NanoPhotometer spectrophotometer. Immediately after RNA extraction, total RNA was reverse transcribed into cDNA following the instructions of the Transcriptor First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany). Later real-time PCR was performed in 25ul reaction system. Samples were denatured at 95°C for 30s, annealed at 60°C for 30s, and extended at 72°C for 40s.The relative mRNA level was determined using the comparative CT method and was normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The primers were synthesized by Sangon Company (Shanghai, China).

The primer sequences for real-time PCR were presented in Table 1.