Stranded RNA-seq Library Preparation

A stranded RNA sequencing library was prepared according to manufacturer’s instructions by using KCTM Stranded mRNA Library Prep Kit (Catalog No. DR08402, Wuhan Seqhealth Co., Ltd., Wuhan, China) (18 (link)). Briefly, the mRNA was enriched by magnetic beads and broken into short fragments by fragment buffer. A random primer was used to synthesize a single strand of cDNA using the mRNA fragment as a template. After synthesizing and purifying double stranded cDNA, the terminus of the cDNA was repaired with base and added with sequencing adaptor. The fragment was caught by magnetic beads and amplified by T100 Thermal Cycler (BIO-RAD, United States). Qubit 3.0 with Qubit RNA Broad Range Assay kit (Life Technologies, Q10210) is used to quantify the cDNA library. Finally, the different cDNA library corresponding to 200–500 bps was sequenced on Illumina Novaseq 6000 sequencer (Illumina Inc., San Diego, CA, United States) with a pair-end 150 bp (PE150) model at Seqhealth Technology Co., LTD (Wuhan, China). The RNA-seq data were obtained based on two independent biological replicates. Each group includes two samples and all the samples were repeated for two times.

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Wu K.J., Liu P.P., Chen M.Y., Zhou M.X., Liu X., Yang Q., Xu L, & Gong Z. (2022). The Hepatoprotective Effect of Leonurine Hydrochloride Against Alcoholic Liver Disease Based on Transcriptomic and Metabolomic Analysis. Frontiers in Nutrition, 9, 904557.

Publication 2022

T100 Thermal Cycler Qubit 3.0 with Qubit RNA Broad Range Assay kit Illumina Novaseq 6000 sequencer

Assay Biological Buffer Cdna Cdna library Mrna Primer Rna seq

Corresponding Organization : Wuhan Polytechnic University

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Variable analysis

independent variables

Use of KCTM Stranded mRNA Library Prep Kit (Catalog No. DR08402, Wuhan Seqhealth Co., Ltd., Wuhan, China) for library preparation

dependent variables

RNA-seq data obtained based on two independent biological replicates
Each group includes two samples and all the samples were repeated for two times

control variables

Magnetic beads used for mRNA enrichment
Fragment buffer used to break mRNA into short fragments
Random primer used to synthesize single-strand cDNA
Double-stranded cDNA synthesis and purification
Terminus repair and adapter ligation
CDNA library fragment size selection (200-500 bps)
Sequencing on Illumina Novaseq 6000 sequencer with pair-end 150 bp (PE150) model
Qubit 3.0 with Qubit RNA Broad Range Assay kit used for cDNA library quantification

controls

No positive or negative controls were explicitly mentioned in the provided information.

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