title: RNA-Seq of NT Chuanxue3 Pb+FeNPs
design: The raw data from sequencing is filtered, and the filtered clean reads are aligned to the reference sequence. Based on the alignment results, novel transcript prediction, SNP and indel detection, alternative splicing, and timing analysis are performed. Quantitative analysis of known and novel genes is also performed, and differential expression analysis is performed based on gene expression levels in different sample groups. Further in-depth analysis is performed on the identified differentially expressed genes, including GO functional analysis, pathway functional analysis, cluster analysis, protein interaction network analysis, and prediction of transcription factor coding capacity                                                                                                                                 1.1 Quality control    Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data(clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30, GC-content and sequence duplication level of the clean data were calculated. All the downstream analyses were based on clean data with high quality.1.2 Comparative analysis    The adaptor sequences and low-quality sequence reads were removed from the data sets. Raw sequences were transformed into clean reads after data processing. These clean reads were then mapped to the reference genome sequence. Only reads with a perfect match or one mismatch were further analyzed and annotated based on the reference genome. Hisat2 tools softwere used to map with reference genome.