Hiseq 2000 system

Total RNA was extracted from ovary tissue at different stages using TRIzol (Invitrogen Carlsbad, CA, USA) according to the manufacturer’s instruction.
Low-molecular weight RNAs (<40 nucleotides long) were isolated from total RNA using a FlashPAGE fractionator (Ambion, Life Technologies, Paisley, UK). miRNA libraries were constructed using the Illumina TruSeq Small RNA Sample Preparation kit, in which miRNAs were ligated to two adapters (5′ Adapter: 5′-GUUCAGAGUUCUACAGUCCGACGAUC-3′; 3′ Adapter: 5′-TGGAATTCTCGGGTGCCAAGG-3′) and amplified by RT-PCR, the amplification products (140–160 bases, including the small RNA and adapter sequences) were further purified on a 15% polyacrylamide TBE gel and sequenced with an Illumina Hiseq 2000 system. The mRNA libraries for different reproductive stages were generated using Illumina Truseq RNA Sample Preparation Kits. The required fragments were enriched by PCR amplification and purified using a Qiagen MiniElute PCR Purification Kit. The library products were sequenced with an Illumina Hiseq 2000 system. The library construction and sequencing of miRNAs and mRNAs was performed at Beijing Institute of Genomics, Chinese Academy of Sciences (Beijing, China).

Whole-genome shotgun sequencing was performed using the Illumina HiSeq 2000 system. Genomic DNA was extracted from fresh mature leaves of the D. longan “HHZ” cultivar using the modified SDS method. DNA sequencing libraries were constructed according to the standard Illumina library preparation protocols. A total of 12 paired-end sequencing libraries, spanning 170, 250, 500, 800, 2000, 5000, 10 000, 20 000, and 40 000 bp, were constructed and sequenced on an Illumina HiSeq 2000 system. After stringent filtering and correction steps using K-mer frequency-based methods [21 (link)], a total of 121.68 Gb of data was obtained and then assembled using SOAPdenovo and SSPACE software [63 (link)]. To check the completeness of the assembly, a longan transcriptome assembly comprising 68 925 unigenes (SRA050205) was mapped to the genome assembly using BLAT32 with various sequence homology and coverage parameters. The BUSCO pipeline [27 (link)] was also used to check the genome completeness.

The total RNA isolated from the normal and mutant capitula was used for Illumina sequencing on an Illumina HiSeq™ 2000 system (Illumina, San Diego, CA, USA). We purified the poly(A) mRNAs, fragmented them into small pieces, and then synthesized the first- and second-strand cDNAs.
The double-stranded cDNAs were purified and resolved for repairing ends and adding a poly(A) tail. Sequencing adapters were then annealed to the short fragments. Briefly, a cDNA library with average insert sizes of 300–500 bp was created and sequenced using the Illumina HiSeq™ 2000 system to generate 100 bp paired-end reads.

The transcriptomic data of GBM patients were obtained from two independent data portals (CGGA and TCGA). As part of the CGGA project, Zhao et al.^{46 (link)} provided the RNA-seq transcriptomic profiles of 325 gliomas samples. The raw RNA-seq data were downloaded from the NCBI Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra) using the accession numbers SRP027383 and SRP091303. The raw fastq files were converted from SRA using Fastq-dump app (version: 2.8.1), and the sam and read count data were obtained by the hisat2 (version 2.1.0) and htseq software (version 0.10.0), respectively. A total of 137 GBM samples with clinical data were selected from the CGGA cohort. Data of an independent cohort of 158 patients was obtained from the TCGA database. The transcriptomic profiles (level 3 data) and corresponding clinical data of these patients were downloaded from the Data Coordinating Center. All RNA-seq libraries were sequenced using the Illumina HiSeq2000 Systems. The raw count data were normalized by the trimmed mean of M-values (TMM) method^{47 (link)}, and genes with extremely low total abundance (count per million <1) were filtered out. The data preprocessing was performed using edgeR package^{48 (link)} (version 3.20.9) in Bioconductor.