Total RNA was extracted from RAW264.7 cells with TRIzol Reagent (Life Technologies, California, USA) according to the instructions. RNA integrity and concentration were determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA, USA). The cDNA library was constructed with the NEBNext Ultra RNA LibraryPrep Kit for Illumina (NEB, E7530) and NEBNext Multiplex Oligos for Illumina (NEB, E7500) according to the manufacturer’s instructions. The cDNA libraries were loaded on an Illumina Nova 6000 sequencing platform at Biomaker (Beijing, China). The adapters and reads from the raw reads of each sample were trimmed to obtain clean reads. The clean reads were mapped against Mus musculus.GRCm38 using HISAT2 v2.0.4. Gene expression levels were estimated using fragments per kilobase of exon per million fragments mapped (FPKM). The false discovery rate (FDR) control method was used to identify the threshold of the P value in multiple tests to compute the significance of the differences. Significant differential expression was accepted as |log2FC|>1.5 and P value<0.05. Functional annotation and enrichment analysis of the significantly differentially expressed genes was performed with the bioinformatic pipeline tool BMK Cloud online platform.
Nova 6000 sequencing platform
Manufactured by Illumina
Sourced in China, United States
The Nova 6000 is a high-throughput DNA sequencing platform designed for large-scale genomic research. It utilizes advanced sequencing-by-synthesis technology to generate massive amounts of sequence data with high accuracy and speed. The Nova 6000 is capable of producing up to 6 billion sequencing reads per run, making it a powerful tool for applications such as whole-genome sequencing, transcriptomics, and metagenomics.
Automatically generated - may contain errors
2 protocols using nova 6000 sequencing platform
1
Transcriptomic Analysis of RAW264.7 Cells
2
DNA Extraction and Sequencing of A. japonicus
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The TIANGEN amp Marine Animals DNA Kit (TIANGEN, Beijing, China) was used for the extraction of total genomic DNA in this experiment. A. japonicus muscle was selected instead of the body wall tissue, to avoid the influence of complex organic substances in the body wall, which might affect the quality of nucleic acids. The purity and integrity of the nucleic acids were initially checked by 1% agarose gel electrophoresis, and samples with clear electrophoretic bands and no or mild degradation were qualified. Qualified DNA samples were adjusted to 100 ng/μl and stored at -20 °C.
Qualified DNA samples were randomly fragmented into 350 bp fragments using a Covaris E220 ultrasonic DNA fragmentation machine (Shanghai Tusheng Vision Technology Co., Ltd., Shanghai, China). The library preparation process was completed on a T100 Thermal Cycler PCR (BIO-RAD, USA) instrument using KAPA HyperHlus reagent (Illumina, USA) after 10 cycles of end repair, addition of polyA tails, addition of sequencing adapters, and purification and amplification. High-throughput sequencing of samples was performed using the Illumina Nova6000 sequencing platform (Illumina, California, USA) with a 150 bp paired-end reads (LC-Bio, Hangzhou, Zhejiang Province, China).
Qualified DNA samples were randomly fragmented into 350 bp fragments using a Covaris E220 ultrasonic DNA fragmentation machine (Shanghai Tusheng Vision Technology Co., Ltd., Shanghai, China). The library preparation process was completed on a T100 Thermal Cycler PCR (BIO-RAD, USA) instrument using KAPA HyperHlus reagent (Illumina, USA) after 10 cycles of end repair, addition of polyA tails, addition of sequencing adapters, and purification and amplification. High-throughput sequencing of samples was performed using the Illumina Nova6000 sequencing platform (Illumina, California, USA) with a 150 bp paired-end reads (LC-Bio, Hangzhou, Zhejiang Province, China).
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