Vahts total rna seq library preparation kit

Total RNA was extracted from TMZ-sensitive/resistant U87MG cell lines by TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RNA quality was assessed by Nanodrop2000 and Qubit 3.0. RNA integrity was determined by Agilent 2100 Bioanalyzer. mRNA Capture Beads (Vazyme Biotech, Nanjing, China) were used to eliminate rRNAs and a VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech) was used to prepare libraries. Sequencing was performed on the Illumina Hiseq 2500 platform (pair-end 150 bp). The raw sequencing data have been deposited in the NCBI (BioProject accession: PRJNA768121). Data were further processed by R (version 4.1.0). A heatmap of gene expression profiles was generated using the pheatmap package. Principal component analysis (PCA) of each sample was performed and the top two principal components were shown. Differentially expressed genes (DEGs) were identified using the limma package (| log2(fold change) | > 1 and adjusted p-value < 0.05 as threshold). A volcano plot was illustrated to visualize the distribution of DDR genes using the EnhancedVolcano package.

Total RNA was extracted from temozolomide (TMZ) sensitive/resistant U87MG cell lines using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was assessed using Nanodrop2000 and Qubit 3.0. RNA integrity was determined by Agilent 2100 Bioanalyzer. Then, total RNA was treated with mRNA Capture Beads (Vazyme Biotech, Nanjing, China) to eliminate rRNAs. A VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech) was used for library preparation. RNA sequencing was performed on the Illumina Hiseq 2500 platform. Pair-end reads were generated with reading lengths up to 150 bp. Differentially expressed genes were identified by limma package in R (logFC > 1 and P < 0.05 as threshold). The heatmap was illustrated to visualize the levels of differential expressed DNA replication-related genes. The volcano plot showed the differential distribution of DNA replication and repair-related genes. The raw sequencing data have been deposited in the NCBI under BioProject accession number PRJNA768121.

For gene expression analysis of the candidate region, blades from two parents living in the same condition were sampled for RNA-seq. Each parent sampling was conducted using three biological replicates. Total RNA was extracted using a Plant RNA Kit (OMEGA), and the first-strand cDNA was prepared using a HiScript II Q RT SuperMix for the qPCR kit (Vazyme Biotech). RNA quality was assessed using an RNA Nano 6000 Assay Kit and Bioanalyzer 2100 system (Agilent Technologies). Sequencing libraries were generated using a VAHTS Total RNA-Seq Library Preparation Kit (Vazyme Biotech). Then, the libraries were paired-end sequenced on an Illumina HiSeq 2000 platform (Illumina, United States) with the read length of 150 bp.
The quality of raw sequencing reads was evaluated using FastQC (Brown et al., 2017 (link)) and Trimmomatic (Bolger et al., 2014 (link)). Differential gene and transcript expression analysis of RNA-seq experiments were performed with TopHat and Cufflinks, respectively (Trapnell et al., 2012 (link)).