RNA-Seq of mus musculus: strawberry leaves
Raw sequencing data was first filtered by fastp (v0.23.2), low-quality reads were discarded and the reads with adaptor sequences were trimmed. Clean Reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. In brief, clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-clusters. After these steps, any errors and biases introduced by PCR amplification or sequencing were eliminated.The sequences were used for standard mRNA-seq analysis. They were mapped to the reference genome using STAR software (version 2.7.6a). Reads mapped to the exon regions of each gene were counted by featureCounts (Subread-1.5.1; Bioconductor) and then RPKM was calculated. Differentially expressed genes between groups were identified using the edgeR package (version 3.40.2), and a p-value cutoff of 0.05 and a fold-change cutoff of 2 were used.