TRIzol reagent (Invitrogen) was used for RNA extraction, and cDNA libraries were prepared based on the MGIEasy RNA Directional Library Prep Kit Guide (MGI Tech, Cat. 1000006385). Equal concentrations of each library were sequenced using a DNBSEQ-G400RS (MGI Tech) platform to create pair-end 100-bp reads. Quality assessment and trimming of the generated sequences were done by the RNA-seq alignment tool from MGI Tech, followed by alignment to the mouse reference genome (mm10). The expression levels of genes in each sample and the corresponding fold changes were estimated by Partek Genomics Suite with GENCODE annotation. Relative expression of each gene is represented by counts per million (CPM). Partek Genomics Suite and statistical package were used for the statistical analysis, hierarchical clustering differential expression analysis.
Dnbseq g400rs
Manufactured by MGI Tech
Sourced in China
The DNBSEQ-G400RS is a next-generation sequencing instrument designed for high-throughput DNA and RNA sequencing. It utilizes the proprietary DNBSEQ technology to generate high-quality sequencing data. The core function of the DNBSEQ-G400RS is to perform massively parallel sequencing of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (1 mentions)
Agilent bioanalyzer device, by Agilent Technologies (1 mentions)
Mgi easy universal library conversion kit, by MGI Tech (1 mentions)
Smart seq ht kit, by Takara Bio (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Ingenuity pathway analysis software, by Qiagen (1 mentions)
Fastgene rna premium kit, by Nippon Genetics (1 mentions)
5 protocols using dnbseq g400rs
1
Transcriptome Profiling of Mouse Samples
2
RNA-seq Analysis of HuH-7 Cells
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HuH-7 cells were transfected with control, FOXA1, or CEBPA siRNAs (10 nM) for 48 h. Total RNA was extracted from the cells using the RNeasy Plus Mini kit. Agilent Bioanalyzer device (Agilent Technologies) was used to assess the quality of extracted RNA. Subsequently, RNA-seq libraries were prepared with total RNA (100 ng) using an NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocol. The libraries were sequenced with 100 bp paired-end reads on the DNBSEQ-G400RS (MGI Tech, Shenzhen, China). The analysis of sequencing data was performed by a standard RNA-seq analytical pipeline. Briefly, STAR(v2.7.3a)45 (link) was used to align the sequencing data to the human genome (hg38). Quantification of gene expression was performed using RSEM (v1.3.3)46 (link). The DESeq2 package (v1.26.0)47 (link) was used to normalize the read count data and test for differential gene expression.
3
PCR-free DNA library preparation and sequencing
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A short-read sequence library was prepared using a PCR-free Swift 2S Turbo Flexible DNA Library Kit (Swift Sciences, Ann Arbor, MI, USA) and converted into a DNA nanoball sequencing library with an MGI Easy Universal Library Conversion Kit (MGI Tech). The library was sequenced on a DNBSEQ G400RS (MGI Tech) instrument in paired-end, 101-bp mode. The obtained reads were used to estimate the genome size with Jellyfish after removing low-quality bases (<10 quality value) with PRINSEQ, adaptor sequences (AGATCGGAAGAGC) with fastx_clipper in FASTX-Toolkit, and reads from organelle genomes (GenBank accession numbers: AP012207 and KY563267 )15 (link),16 by read mapping with Bowtie2 on the reference sequences.
4
RNA-seq Analysis of Differential Gene Expression
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A quality check of the total RNA was performed with Bioanalyzer (Agilent Technologies, California, USA). RNA-seq libraries were synthesized with the SMART-seq HT Kit (634455, Clontech). RNA-seq libraries were sequenced using paired end reads (150 nt for read 1 and 2) on a DNBSEQ-G400RS (MGI Tech, Shenzhen, China). The obtained reads were mapped to the mouse GRCh38.p13 (hg38) genome with STAR software (version 2.7.9). Reads of annotated genes were counted using featureCounts software (version 2.0.1). FPKM values were calculated from mapped reads by normalizing to total counts and the transcript. Differentially expressed genes were detected with the DESeq2 software package (version 1.20.0). A list of differentially expressed genes detected by DESeq2 (basemean >5 and fold change <0.25, or basemean >5 and fold change >4) was used for GO enrichment analysis with the clusterProfiler software package (Yu et al., OMICS 2012, 16:5, version 3.16.0). Pathway analysis was performed using Ingenuity Pathway Analysis software (QIAGEN) and a biological triplicate RNA-seq dataset.
5
RNA-seq analysis of irradiated HMECs
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Total RNA was extracted from HMECs at 24 h post-irradiation by using a FastGene RNA Premium Kit (NIPPON Genetics). To ensure the accuracy of the data, each sample was mixed with total RNA obtained from two independent experiments. RNA quality was checked using agarose electrophoresis (total RNA > 1.0 µg, OD260/280 = 1.8–2.2, RIN > 6.5). RNA-seq was performed via next-generation sequencing using DNBSEQ-G400RS (RRID:SCR_017980; MGI Tech Co., Ltd., Shenzhen, China). All analyses were performed using integrated Differential Expression and Pathway analysis (iDEP) version 0.95 (http://bioinformatics.sdstate.edu/idep95/ ) [29 (link)].
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