Paired-end libraries were prepared using an ABclonal mRNA-seq Lib Prep Kit (ABclonal, China), following the manufacturer’s instructions. The mRNA was purified from 1 μg of total RNA using oligo (dT) magnetic beads, followed by fragmentation using divalent cations at elevated temperatures in ABclonal First Strand Synthesis Reaction Buffer. Subsequently, first-strand cDNAs was synthesized with random hexamer primers and reverse transcriptase (RNase H), using mRNA fragments as templates, followed by second-strand cDNA synthesis using DNA polymerase I, RNase H, buffer, and dNTPs. The synthesized double-stranded cDNA fragments were then adapter-ligated to prepare the paired-end library. Adaptor-ligated cDNA was used for PCR amplification. PCR products were purified (AMPure XP system) and library quality was assessed using an Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an Illumina Novaseq 6000 (or MGISEQ-T7) and 150 bp paired-end reads were generated. Data generated from the Illumina (or BGI) platform were used for bioinformatics analysis. The raw data were deposited in the NCBI Sequence Read Archive (SRA) database with accession number PRJNA898498.
Mgiseq t7
Manufactured by Illumina
The MGISEQ-T7 is a next-generation sequencing (NGS) platform designed for high-throughput DNA and RNA sequencing. It utilizes sequencing-by-synthesis (SBS) technology to generate high-quality sequencing data. The MGISEQ-T7 offers a range of flexible configurations to accommodate various sequencing applications and throughput requirements.
Automatically generated - may contain errors
4 protocols using mgiseq t7
1
Paired-end RNA-seq Library Preparation
2
Transcriptome Analysis via mRNA-seq
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RNA was extracted from 1 µg of samples following the standard protocol of Shanghai Applied Protein Technology. The RNA quality was assessed using a Nanodrop ND-2000 system and an Agilent Bioanalyzer 4150 system. A mRNA-seq library prep kit was used to prepare the samples for transcriptome sequencing. mRNA was extracted from the total RNA using oligo magnetic beads. First and second strand cDNA were synthesized from the fragmented RNA using reverse transcriptase, DNA polymerase, and RNase H. The double stranded cDNA fragments were prepared for PCR amplification. A final cDNA library was obtained after PCR enrichment. The library was sequenced on an Illumina Novaseq 6000 or MGISEQ-T7 platform, generating 150 bp paired-end reads.
The sequencing data generated on the Illumina or BGI platform was used for bioinformatics analysis. Clean data was obtained by removing low quality reads, reads containing adapters, and poly-N reads using in-house scripts. Whole procedure was conducted through the FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ).
The sequencing data generated on the Illumina or BGI platform was used for bioinformatics analysis. Clean data was obtained by removing low quality reads, reads containing adapters, and poly-N reads using in-house scripts. Whole procedure was conducted through the FastQC tool (
3
High-Quality Gut Microbiome Genome Sequencing
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All the isolates were grouped into species-level clusters based on a threshold of 98.7% identity of the 16S rRNA gene sequence47 . Our strategy for selecting strains for WGS were (1) representation of candidates for unidentified taxa, (2) covering as many taxa as possible of the strains cultivated in the study, (3) important species from various donors. Among the 1804 newly sequenced strains, 1282 were sequenced using the MGISEQ-T7 platform, and the remaining 522 were sequenced by the Illumina Hiseq 2000 platform. The methods of whole-genome sequencing and de novo assembly were as described by Zou et al.9 (link). Gene numbers were calculated using GeneMarkS-2 (v1.10)48 (link). Genome quality was evaluated using CheckM (v1.1.2)49 (link) ‘lineage_wf’ workflow to select genomes with >90% completeness and <10% contamination as high-quality genomes. The gene prediction and enzyme annotation of the 3324 genomes were performed on Prokka 1.14.650 (link).
4
Sirt3-Mediated Transcriptome Profiling in BMDMs
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The total RNA is isolated in BMDMs of Sirt3+/+ and Sirt3−/− treated with C16:0 + MSU in triplicate. Transcriptome sequencing and subsequent bio-informatics analysis were carried out by the Applied Protein Technology Co., Ltd. (Shanghai, China). The mRNA was purified from 1 μg total RNA using oligo (dT) magnetic beads followed by fragmentation in the ABclonal First Strand Synthesis Reaction Buffer. Subsequently, using mRNA fragments as templates, the first strand of cDNA is synthesized using random primers and Reverse Transcriptase (RNase H), followed by the second strand of cDNA synthesis using DNA polymerase I, RNAseH, buffer, and dNTPs. The synthesized double-stranded cDNA fragments are ligated to the linker sequence for PCR amplification. The PCR product was purified and library quality was evaluated using Agilent Bioanalyzer 4150 system. Finally, the library preparations were sequenced on an Illumina Novaseq 6000 (or MGISEQ-T7), and 150 bp paired-end reads were generated. The data generated from Illumina (or BGI) platform were used for bioinformatics analysis. Differential expression genes (DEGs) analysis was performed using the DESeq2, DEGs with | log2FC |> 1 represent upregulation, DEGs with | log2FC |< − 1 mean downregulation, and Padj < 0.05 were considered to be significantly differentially expressed genes.
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