Hiseq pe150 sequencing

Based on simplified genome sequencing, the qualified DNA was digested with Mse I restriction endonuclease and a linker with a barcode was added to both sides of the enzyme section. Then, PCR amplification was carried out, and the amplified products were mixed. The required fragments were selected to establish the library. The constructed library was initially quantified using Qubit^®2.0 and diluted to 1 ng·μL⁻¹. The library was examined using an Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). After the length of the inserted fragment was obtained, the effective concentration of the library was accurately quantified using q-PCR (the effective concentration of the library > 2 nmol·L⁻¹) to ensure the quality of the library [12 (link)]. The library was qualified. The pool was mixed according to the effective concentration of different libraries and the amount of data required for the target machine, followed by Illumina Hi-seq PE150 sequencing [13 (link)].

The total RNA was extracted from the LINC15957 overexpressed plants and control radish leaves using RNAprep Pure Plant Kit (TIANGEN, Beijing, China). A cDNA library was constructed for each sample using enriched mRNA. The libraries were sequenced using the Illumina HiSeq-PE150 sequencing platform from BeijingNovogene Co. Ltd. Reference genome and gene model annotation files were downloaded from genome website directly (http://39.100.233.196:82/download_genome/Brassica_Genome_data/Rapsa_Xiang_V1.0/). Index of the reference genome was built using Hisat2 v2.0.5 and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5.
Differential expression analysis of two processing samples (three biological replicates per process) was performed using the DESeq2 R package (1.20.0). Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the clusterProfiler R package, in which gene length bias was corrected. GO terms with corrected Pvalue less than 0.05 were considered significantly enriched by differentially expressed genes. ClusterProfiler R package was used to test the statistical enrichment of differential expression genes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways.

Two biological replicate samples from two independent plants of each genotype of MG, Br, and Pink stages have been harvested. Total RNA was extracted using the NucleoSpin RNA Plant kit (Macherey-Nagel) and sent to the Novogene Company (Beijing, China) for Illumina HiSeq PE150 sequencing. The cDNA library was constructed following the manufacturer’s recommendations and then purified to remove the low-quality sequences. The clean data are available in Zenodo (https://zenodo.org/) (doi:10.5281/zenodo.5525948 and 10.5281/zenodo.5525946). The RNA seq data were analyzed using LSTrAP (Proost et al., 2017 (link)). The clean reads of each sample were aligned to the Tomato Genome version SL4.0 and Annotation ITAG4.0 (ftp://ftp.solgenomics.net/tomato_genome/annotation/ITAG4.0_release/). The DEGs between transgenic fruit and WT fruit were identified under the parameter of FC ≥ 2 and FDR < 0.05.