Hiseq 4000 sequencing system

The testis samples from 8-week-old C57BL/6 male mice were washed in 1×phosphate-buffered saline (PBS). Total RNAs were extracted from the samples using the Dynabeads mRNA Purification Kit (61006, Thermo Fisher Scientific). Ribo-Zero™ rRNA Removal Kit (MRZPL1224, llumina) was used to remove rRNA of samples. RNA integrity was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies). An mRNA sequencing library for RNA-seq was constructed using the TruSeq RNA Library Prep kit v2 (RS-122-2001, Illumina), followed by paired-end (2 × 100 bp) sequencing using the Illumina HiSeq 4000 Sequencing System (Illumina) in the Beijing Genomics Institute. Raw paired-end reads were filtered by FASTX-Toolkit. The quality of the reads was confirmed by FastQC (ver:0.11.3). Raw data (raw reads) of fastq format were processed through in-house perl scripts. Feature Counts (ver: l.5.0-p3) was used to count the reads numbers mapped to each gene. FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. Signaling pathway matching analysis for the differentially expressed gene (DEG) list was performed using KEGG Mapper in Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/). The mapping of GO to DEGs was carried out using Blast2GO (ver: 4.1.9).

Total RNA was extracted from frozen uterus tissue sample using TRIzol reagent (TransGen Biotech, Beijing, China)^{71 (link)}. The quality and quantity of RNA were evaluated by 1% agarose gels, NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA), respectively. The RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). 3 μg RNA per sample was prepared to generate individual bar-coded sequencing libraries using NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations. Sequencing of these libraries was performed on an Illumina HiSeq 4000 sequencing system (Illumina, San Diego, CA, USA) using the 150-bp pair-end sequencing strategy, following the manufacturer’s instructions.

Total RNA was isolated from frozen true leaf tissues using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer’s instructions. RNA purification steps and on-column DNase digestion was performed using the QIAGEN RNeasy Mini Kit as suggested by the manufacturer’s instructions (QIAGEN, Hilden, Germany). The total RNA qualities were further analyzed using Agilent RNA 6000 Nano Kit (Agilent Technologies Inc., Santa Clara, CA, USA) ran on a Agilent 2100 Bioanalyzer, with average RNA RIN values > 7. RNA-seq libraries were prepared as described previously^{25 (link)}. The quality and integrity of each library was checked with Agilent high-sensitivity DNA chips (Agilent Technologies, Santa Clara, CA, USA). Concentrations of sequencing libraries were quantified using Qubit fluorometer and Qubit dsDNA HS assay kit (Life Technologies, CA, USA). Three biological replications of each genotype (USVL531-MDR, USVL677-PMS) per time-point were used for 150 bp paired-end RNA-seq sequencing (Duke Center for Genomic and Computational Biology, Durham, NC, USA) with Illumina HiSeq 4000 sequencing system (Illumina, Inc., San Diego, CA, USA).

Total RNA (3 μg per sample) was used to construct each sequencing library. Briefly, ribosomal RNA was removed using the Epicentre Ribo-zero™ rRNA Removal Kit as instructed (Epicentre, Madison, WI, USA), followed by ethanol precipitation. The resulting mRNAs were fragmented using divalent cations under elevated temperature. The short mRNA fragments were then used to synthesize the first-strand cDNA using random hexamers primers. The second-strand cDNA synthesis was performed using DNA polymerase I and RNase H. The cDNA libraries were prepared by using the standard Illumina protocols and then sequenced on an Illumina Hiseq™ 4000 sequencing system (Illumina, San Diego, CA, USA) to generate reads with 150 bp paired-ends. Constructions of libraries and RNA sequencing were performed by the Zhejiang TianKe High-Technology Development Co., Ltd. (Hangzhou, Zhejiang, China).

Following the standard protocol, total RNAs were extracted from the gill and adipose fin tissues of the fish samples in the HT and NT groups using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). RNA quality and integrity were assessed on an Agilent 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) using RNA ScreenTape. RNA concentrations were measured by a Qubit^® 2.0 Fluorometer RNA assay kit (Life Technologies, Carlsbad, CA, USA). Approximately 2 µg of total RNA from each sample was used to construct complementary DNAs (cDNAs) with SuperScript II (Invitrogen, Carlsbad, CA, USA). We prepared cDNA libraries using a TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Index adapters were added to identify sequences for each sample in the final data. The quality of the libraries was measured on an Agilent 2200 Tapestation using High Sensitivity RNA ScreenTape, and qPCR was measured using a KAPA SYBR Fast qPCR Kit (NIPPON Genetics, Tokyo, Japan). Subsequently, the 40 libraries (10 biological replicates per group (HT and NT), per tissue (gill and fin)) were subjected to paired-end (2 × 100 bp) sequencing on the Illumina HiSeq 4000 Sequencing System (Illumina) at BGI, Japan, and all samples were sequenced in the same lane.

Monocytes were cultured with or without 100 µM H₂O₂ for 7 days. Respective total RNAs were extracted and random primers were used to generate cDNA. Sequencing was carried out using an Illumina HiSeq 4000 Sequencing System for 150 cycles (KangChen Bio‐tech Inc, Shanghai, China). After the data preprocessing, fragments per kilobase of exon per million fragments mapped (FPKM) and differentially expressed genes (DEGs) were then calculated,³⁹, ⁴⁰ with the FPKM ≥ 0.5 (Cuffquant) considered statistically significant. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) tool was used for pathway analysis of the DEGs.

The tumor samples collected from mice were washed in 1X PBS. Total RNA was extracted from tumor samples using the Dynabeads mRNA Purification Kit (61006, Thermo Fisher Scientific), and RNA quality was evaluated using the Agilent 2100 Bioanalyzer system (Agilent Technologies). An mRNA sequencing library was constructed, and the Illumina HiSeq 4000 Sequencing System (Illumina) was used for paired‐end sequencing. Signaling pathway annotation analysis of differentially expressed genes (DEGs) was performed with Kyoto Encyclopedia of Genes and Genomes (KEGG, https://www.genome.jp/kegg/).