title: C-1
design: Transcriptome broadly refers to the collection of all transcriptome products in a cell under a certain physiological condition. The research object is the sum of all RNA that can be transcribed by a specific cell under a certain functional state, mainly including mRNA and ncRNA. In a narrow sense, it refers to the collection of all mRNA, and the RNA detected in this project specifically refers to mRNA.Qubit4.0 fluorometer/MD microplate reader was used to accurately measure RNA concentration and qsep400 biological analyzer was used to accurately detect RNA integrity. After the RNA samples passed the test, the mRNA was isolated and the cDNA was synthesized, and then the whole library was prepared through the steps of terminal repair, adding a tail, adding sequencing connector, purification, PCR amplification, denaturation, cyclization and so on. After the construction of the library, qubit was used for preliminary quantification, and then Agilent 2100 was used to detect the inserted fragments of the library to ensure the quality of the library.Qubit2.0 was used for preliminary quantification, Agilent2100 was used to detect the insertsize of the library, and the next step of the experiment could be carried out only after the insertsize met the expectations. The effective concentration of the library was accurately quantified by Q-PCR (the effective concentration of the library was>2nm), and the library inspection was completed. After the library passed the test, DNB (dnananoball) was prepared, and then loaded on the sequencing chip for sequencing using high-throughput sequencer.The original paired end reading is trimmed and passed fast https://github.com/OpenGene/fastp (0.23.2) and default parameters for quality control. Use hisat2 (2.2.1) to perform sequence alignment between cleanreads and the reference genome to obtain the location information on the reference genome or gene, as well as the unique sequence characteristics of the sequenced sample. The BAM file of the alignment results can be viewed through igv[3] software. According to the location information of the reads of the genome on the comparison, the reads are assembled into transcripts using stringtie (2.1.6), and the new transcripts information is extracted from the comparison results of the spliced transcripts and genome annotation. Extract the sequence of the new gene from the genome, and use diamond[5] to compare the new gene with KEGG, go, NR, Swiss prot, TrEMBL, KOG database sequence to obtain annotation results. The alignment condition is evalue1e-5. The clusterprofiler (v4.6.0) program is used to obtain go enrichment analysis of uniquely assembled transcripts to describe biological processes, molecular functions and cellular components. The Kyoto Encyclopedia of genes and genomes (KEGG) was used for metabolic pathway analysis [22]. During this period, gene quantitative analysis and differential expression analysis will be carried out (condition screening | log2foldchange |>=1&&fdr<0.05).After the completion of transcriptome analysis, the combined analysis of metabolome and transcriptome will be carried out, including principal component analysis (PCA), correlation analysis, heat map, network diagram, K-means clustering analysis, canonical correlation analysis (CCA) and O2PLS analysis.