title: RNA-seq of Morchella esculenta.sp
design: according to the manufacturers instructions (TruSeq RNA Sample Preparation Kit, San Diego, CA). Briefly, the RiboZero rRNA removal kit (Epicenter, USA) and Fragmentation Buffer were used for rRNA removal and fragmentation, respectively. cDNA synthesis, end repair, A base addition, and connection of Illumina index connectors were performed according to the Illumina protocol. The library was then sized for 200300 bp cDNA target fragments on 2% agarose, and then amplified by 15 PCR cycles using Phusion DNA polymerase (NEB, USA). Paired-end libraries were sequenced after TBS380 (YPH-Bio, Beijing) quantification using an Illumina NovaSeq 6000 sequencer (150bp*2, Shanghai BIOZERON Co., Ltd ). Trimming and quality control of raw paired-end reads was conducted using the Trimmomatic software package (SLIDINGWINDOW:4:15 MINLEN:75). The Rockhopper software was then used to individually align the clean reads to a reference genome with orientation patterns. Computational analysis of bacterial RNA-seq data was also conducted with Rockhopper using the RNA sequencing reads generated by high-throughput sequencing technology as input. The software calculates gene expression levels using default parameters. The numbers of fragments per kilobase per million mapped reads (FPKM) were then used to calculate the expression level of each transcript to discriminate DEGs (differentially expressed genes) between samples from two different treatments. The edgeR package was used for differential expression analysis. The DEGs between the two samples were selected using the following criteria: the FDR (false discovery rate) should be less than 0.05 and the log fold change greater than 2. The Goatools library was used to perform GO (Gene Ontology) functional enrichment analysis to gain insights into the function of the DEGs, and KOBAS (KEGG Orthology Based Annotation System) was used for KEGG pathway analysis. DEGs were considered to be significantly enriched in GO terms and metabolic pathways when the Bonferroni corrected P-value was less than 0.19.