Comprehensive RNA-Seq Protocol for Transcriptome Analysis

Total RNA was isolated and underwent quality assessment with Agilent 2000 bioanalyzer (Agilent Technologies, Netherlands). The library was generated with NEBNext Ultra II Directional RNA-Seq Kit (New England Biolabs, UK). Next, the mRNA was isolated, fragmented and synthesized using the Poly(A) RNA Selection Kit (Lexogen, Austria) according to the manufacturer’s instructions. The Illumina indexes 1-12 were used for indexing. The raw sequencing data underwent quality control by FastQC(https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). HiSeq X 10 (Illumina, USA) was utilized for high-throughput paired-end 100 sequencing, and the adapter and low-quality reads (< Q20) were excluded by FASTX_Trimmer ((http://hannonlab.cshl.edu/fastx_toolkit/) and BBMap ((https://sourceforge.net/projects/bbmap). For analysis, the reads were mapped to the reference genome via TopHat [23 (link)]and the expression levels were calculated based on the Fragments Per Kilobase Million (FPKM) mapped reads from Cufflinks [24 (link)]. Quantile normalization was performed via EdgeR (R Development Core Team, 2016) and ExDEGA (E- biogen, Inc., Korea) was used for mining and graphic visualization of the data. A more detailed description of the method is provided in our previous study [4 (link)].

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Jang H.J., Lee S., Hong E., Yim K.J., Choi Y.S., Jung J.Y, & Kim Z.H. (2022). Mychonastes sp. 246 Suppresses Human Pancreatic Cancer Cell Growth via IGFBP3-PI3K-mTOR Signaling. Journal of Microbiology and Biotechnology, 33(4), 449-462.

Publication 2022

Agilent 2000 bioanalyzer NEBNext Ultra II Directional RNA-Seq Kit Poly(A) RNA Selection Kit HiSeq X 10

Library Mrna Poly a rna Rna seq

Corresponding Organization :

Other organizations : Korea Research Institute of Bioscience and Biotechnology, Korea Basic Science Institute, National Institute of Biological Resources, CHA University

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Variable analysis

independent variables

Total RNA isolation
Library generation with NEBNext Ultra II Directional RNA-Seq Kit
MRNA isolation, fragmentation, and synthesis using the Poly(A) RNA Selection Kit
Illumina index usage for indexing

dependent variables

Sequencing data quality assessment by FastQC
Adapter and low-quality reads (< Q20) exclusion by FASTX_Trimmer and BBMap
Reads mapping to reference genome via TopHat
Expression levels calculation based on FPKM mapped reads from Cufflinks
Quantile normalization via EdgeR and data mining/visualization using ExDEGA

control variables

Manufacturer's instructions followed for library preparation and sequencing

positive controls

None specified

negative controls

None specified

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