Hiseq xten novaseq 6000 sequencer

An RNA sequencing library was prepared using the TruSeq™ RNA Sample Preparation Kit from Illumina (San Diego, CA) following the manufacturer’s recommendations. In brief, mRNA was purified via Poly (A) selection with oligo(dT) cellulose. and then fragmented in fragmentation buffer. Continually, double-stranded cDNA was synthesized using a Super Script Double-Stranded cDNA Synthesis Kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA was subjected to end-repaired, phosphorylation and the A-tailed according to library-construction protocol of Illumina. Libraries were size-selected for cDNA target fragments of 300 base pairs (bp) on a 2% low-range ultra agarose gel, followed by PCR amplification. Finally, the amplified fragments were sequenced with an Illumina HiSeq Xten/NovaSeq 6000 sequencer according to the manufacturer’s instructions^{21 (link)}. The sequencing data have been deposited in the NCBI Sequence Read Archive (SRA), and are accessible through the accession number PRJNA727442.

A transcriptome sequencing (RNA-seq) transcriptome library was prepared with a TruSeq RNA sample preparation kit from Illumina (San Diego, CA) using 1 μg of total RNA. Briefly, mRNA was isolated according to the poly(A) selection method with oligo(dT) beads and then fragmented by fragmentation buffer. Next, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). The synthesized cDNA the was subjected to end repair, phosphorylation, and “A” base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra agarose, followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, the paired-end RNA-seq library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150-bp read length). The raw paired-end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Clean reads (see Table S2 in the supplemental material) then were aligned to the reference genome of Candida glabrata CBS 138 (https://www.ncbi.nlm.nih.gov/genome/192?genome_assembly_id=28426). The differential expression analysis was performed using the DESeq software.

Transcriptome assay was carried out by Meiji Biotech (Shanghai, China). Briefly, total RNA was extracted from the tissue using TRIzol® Reagent according to the manufacturer’s instructions (Invitrogen), and genomic DNA was removed using DNase I (TaKara). Then, RNA quality was determined using 2100 Bioanalyser (Agilent), and RNA was quantified using the ND-2000 (NanoDrop Technologies). RNA-sequencing transcriptome library was prepared using TruSeqTM RNA sample preparation kit from Illumina (San Diego, CA, USA) with 1 μg of total RNA. After quantification by TBS380, paired-end RNA sequencing library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length).

RNA-seq transcriptome libraries were prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA, United States) using 1 μg of total RNA. Firstly, messenger RNA was isolated according to polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer. Secondly, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, United States) with random hexamer primers (Illumina). Then the synthesized cDNA was subjected to end-repair, phosphorylation and “A” base addition according to Illumina’s library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplified using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantified by TBS380, paired-end RNA-seq sequencing library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length). Statistical methods were used to calculate the base distribution and quality fluctuation of each cycle of all sequenced reads, which could intuitively reflect the library construction quality and measurement of sequenced samples from a macro-perspective.

RNA-extract and RNA-seq of muscle are conducted according to standard procedures of Majorbio with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2×150bp read length). The raw paired end reads were trimmed and quality controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) with default parameters. Then clean reads were separately aligned to reference genome with orientation mode using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) software (8 (link)). The mapped reads of each sample were assembled by StringTie (https://ccb.jhu.edu/software/stringtie/index.shtml?t=example) in a reference-based approach (9 (link)). To identify DEGs (differential expression genes) between two different samples, the expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. RSEM (http://deweylab.biostat.wisc.edu/rsem/) (10 (link)) was used to quantify gene abundances. Essentially, differential expression analysis was performed using the DESeq2 (11 (link))/DEGseq (12 (link))/EdgeR (13 (link)) with Q value ≤ 0.05, DEGs with |log2FC|>1 and Q value ≤ 0.05(DESeq2 or EdgeR)/Q value ≤ 0.001(DEGseq) were considered to be significantly different expressed genes. The transcriptomic sequence data have been deposited in the NCBI database (Accession No. PRJNA898816).

The liver and intestine tissues (duodenum) of experimental mice were isolated and stored at -80°C until use. Then the frozen tissues (50 mg/sample) were send for RNA-extraction and RNA-seq analysis (Majorbio Bio-pharm Technology Co.,Ltd, Shanghai, China). Briefly, total RNA was extracted from different groups with three replicates. RNA-seq transcriptome library was prepared with 1 μg of total RNA using TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA). Then the paired-end RNA-seq library was sequenced with the Illumina HiSeq xten/NovaSeq 6000 sequencer (2 × 150 bp read length). The raw paired-end reads were trimmed and quality was controlled by SeqPrep (https://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle), followed by aligning to the reference genome with orientation mode using HISAT2 software (57 (link)). The mapped reads of each sample were assembled by StringTie in a reference-based approach (58 (link)). The expression level of each transcript was calculated according to the transcripts per million reads (TPM) method. The TPM value of each DEG was shown in supplementary files. Transcript abundances were quantified using RSEM software tool (59 (link)). DEGs analysis was performed by DESeq2 with adjusted P value ≤ 0.05. KEGG pathway analysis were performed using KOBAS (http://kobas.cbi.pku.edu.cn/home.do).