Dna clean beads

The sequencing procedures were performed as previously reported [19 (link)] with minor modifications. Briefly, PCR amplification of the bacterial 16S rRNA gene V3–V4 region was carried out using the forward primer 338F (5′-ACT CCT ACG GGA GGC AGC A-3′) and reverse primer 806R (5′-GGA CTA CHV GGG TWT CTA AT-3′). Sample-unique barcodes were integrated into the forward primers to identify sequences from different samples. The PCR system contained 0.25 μL of DNA Polymerase (ABclonal, Wuhan, China), 5 μL of 5 × reaction buffer, 5 μL of 5 × high GC buffer, 2 μL of 2.5 mM dNTP, 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 2 μL of DNA template and 8.75 μL of ddH₂O. The PCR program began with an initial denaturation for 5 min at 98 °C, followed by 25 cycles of denaturation (30 s at 98 °C), annealing (30 s at 52 °C) and extension (45 s at 72 °C) with a final extension for 5 min at 72 °C. PCR products were purified with DNA clean beads (Vazyme, Nanjing, China) and quantified using a fluorescence assay kit (Invitrogen, Carlsbad, CA, USA). Next, the library was constructed using a DNA library preparation kit (Illumina, San Diego, CA, USA) and quantified using a fluorescence assay kit. Finally, sequencing data were produced using a NovaSeq reagent kit (Illumina) by 2 × 250 bp paired-end sequencing on the NovaSeq platform at Personal Biotechnology (Shanghai) Co., Ltd.

Libraries of rProbe protocol were prepared with VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing, China) according to the manufacturer’s protocol. Briefly, the purified RNA was fragmented into short fragments of 250–300 nt using fragmentation buffer and reverse transcribed into cDNA with random hexamer primers. The second strands were synthesised with dUTP for the strand-specific RNA-seq library. Samples were mixed with adaptors, respectively, for ligation and amplification. Final PCR products were purified with 0.9 × DNA Clean Beads (Vazyme, Nanjing, China) and sequenced on Illumina HiSeq Xten platform (Annoroad, Beijing, China) using paired-end sequencing (2 × 150 bases). About 60 to 90 million pairs of reads were generated from each library (n = 6).

The target region was first amplified with barcoded primers (first-round PCR, PCR1) using the Phanta Max Super-Fidelity DNA Polymerase (P505, Vazyme). Then, the PCR1 products were pooled with equal amount and purified by gel extraction or exonuclease I digestion to remove traces of barcoded primers, followed by the second-round PCR (PCR2) using index primers (N321/N322, Vazyme). Libraries were purified using DNA Clean Beads (N411, Vazyme) for sequencing using Illumina NovaSeq platform. Barcoded primers used for PCR1 are listed in table S2. For data analysis, the trimmed reads were aligned to the mitochondrial genome reference sequence of Rattus norvegicus (NC_001665) using bowtie2 with default parameters. The alignment results were converted to bam format by Samtools. Samtools mpileup was used to detect C-to-T or G-to-A conversion. Sites with conversion rate ≥ 1% in any untreated sample were identified as SNP and excluded for further analysis.

The 3 μg of RNA per sample was used as the input material for RNA sample preparation. mRNA was separated from total RNA and purified by mRNA Capture Beads (Vazyme, Nanjing, China). The purified mRNA was incubated in a preheated Polymerase chain reaction (PCR) instrument (Life Technologies, CA, USA) at 94°C for 7 min, interrupted, cooled immediately after the interruption, and centrifuged. The first strand of cDNA was synthesized using the supernatant containing fragment RNA as the template. The double‐strand synthesis reaction reagents were added to the first strand product of the synthesized cDNA, mixed, centrifuged, and incubated in a metal bath to synthesize the second strand. Magnetic beads were used to purify the double‐strand cDNA product. After adapter ligation, the fragments were screened by DNA clean beads (Vazyme, Nanjing, China), and the library was enriched by PCR using the screened cDNA fragments as templates.
Qubit 3.0 fluorescence quantifier (Thermo Fisher Scientific, Waltham, MA, USA) was used for preliminary quantification until the concentration reached more than 1 ng/μL. The Qsep400 high throughput analysis system was used to detect the inserted fragments of the library. After the pieces were inserted as expected, Q‐PCR was used to accurately quantify the effective concentration of the library (effective concentration>2 nM).