Hiseq x10

Thirty-four RNA samples collected were sent for sequencing. Seventeen of these samples were extracted and matched to healthy controls from patients with HCC. Based on previous studies, the sequencing library was prepared after the removal of rRNA according to the Illumina TruSeq RNA sample preparation guide (Illumina, San Diego, California, USA). The index adaptor was ligated once the double-stranded cDNA had been synthesized. After size selection using Agencourt AMPure XP (Beckman), Qubit 2.0 Fluorometer with Qubit dsDNA HS Analysis Kit (Invitrogen, Eugene, OR, USA) and Agilent Bioanalyzer Quantitative and qualitative library (Agilent Technologies, Santa Clara, CA, USA), samples were submitted to Illumina HiSeq X-10 (Illumina, San Diego, CA, USA) for pair-end sequencing of 2 × 150 bp.

The whole genomic DNA of M.matsumurae was sequenced using next-generation sequencing (Illumina HiSeq X10, Biomarker Technologies Corporation, Beijing, China). About 2.13 Gb clean data were assembled into a complete circular mitogenome using NOVOPlasty v.4.3.1 (Dierckxsens et al. 2017 (link)) with the cox1 gene of Saissetiacoffeae (Lu et al. 2020 (link)) as the seed sequence.

The aliquots of livers from three randomly selected rats from the normal diet, HCHFD, and HCHFD+HTE groups were ground into powder in liquid nitrogen. Total RNA was extracted from using Trizol reagent (Invitrogen) according to the manufacturer’s instructions. To avoid genomic DNA contamination, total RNA samples were treated with a RNase-Free DNase Kit (Invitrogen) following the manufacturer’s instructions. The quality of RNA was evaluated using Agilent 2100 BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). The purified RNA then was used to synthesize double-strand complementary DNA (cDNA). The cDNA libraries were quantified using the Kapa Library Quantification Kit (Kapa Biosystems, Boston, MA, USA). After cluster amplification of the denatured templates, samples in flow cells were sequenced using the Illumina HiSeq X10 (Illumina) with the strategy of PE50.
StringTie (version 1.3.3b) was used to align transcript sequences obtained from RNA-seq to the UCSC reference genome rn6 and to estimate the transcript levels of genes. Differentially expressed genes were identified using DESeq2 (version 1.16.1) with the cutoff at fold change ≥2 and p value ≤0.05.

Blood samples of each KD patient were collected by the EDTA anticoagulation tube. DNA was extracted using Genomic DNA Extraction Kit (Cat. No.DP329, TIANGEN Bioscience Beijing), and the library was constructed using KAPA HTP Library preparation kit (Cat. No. KK8234, Roche, USA). Illumina HiSeq X10 (Illumina USA) was used as the sequencing platform for libraries target enriched by custom capture array. The hybridization probes of the custom capture array consisted of previous published 472 GWAS hotspots related to KD and 560 candidate genes among 4 KEGG pathways including Toll-like receptor signaling pathway, Cytokine receptor interaction, TGF-β signaling pathway and T cell receptor signaling pathway. The targeted capture chips were designed by SeqCap EZ Choice (Roche, Switzerland).

RNA-seq and analysis were conducted as previously described (Huang et al., 2021 (link)). Total RNA was extracted from whole cells with the mirVana miRNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA quality and integrity were evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies). Samples with an RNA integrity number ≥7 were considered to be of high quality and were processed further and subjected to subsequent analysis. Total RNA-seq libraries were generated using 4 μg of total RNA, which was analyzed using the TruSeq Stranded mRNA LTSample Prep Kit (Illumina). These libraries were then sequenced using the Illumina sequencing platform (HiSeqTM 2500 or Illumina HiSeq X 10), and 125-bp and 150-bp paired-end reads were generated. Transcriptome sequencing was conducted by OE Biotech Co., Ltd., and clean reads were provided. The clean reads were mapped to the hg38 reference genome using hisat2 (version 2.1.0). The output BAM files were converted to SAM files using SAMtools 1.9. The final transcripts per million (TPM) values were obtained using Stringtie 1.3.5.

The cDNA libraries were prepared by NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, USA, #E7760S) following manufacturer’s recommendations. Briefly, the first strand cDNA was synthesized using random primers and M‐MuLV Reverse Transcriptase (RNaseH^‐) after fragmentation of the RNAs. Then, the second strand cDNA was synthesized with DNA polymerase I and RNase H, and also, dUTP was introduced in this step. Subsequently, remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3ʹ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. 150–200 bp of cDNA fragments were enriched in the following size selection step by using AMPure XP beads system (Beckman Coulter, Beverly, USA). Then, the libraries were digested with 3 μL USER Enzyme (NEB, USA) at 37°C for 15 minutes. Then, preamplification was performed with Phusion High‐Fidelity DNA polymerase, and Index was introduced in this step. Finally, the PCR products were purified and cDNA library concentration was assessed using a Qubit® 2.0 fluorometer. The library was sequenced by Illumina HiSeq X-10 (Illumina Inc., San Diego, CA, USA) sequencer using a 2 × 150 bp paired-end pattern (PE150).