Hiseq 4000 system

The whole-genome sequencing was implemented using an Illumina HiSeq 4000 system (Illumina, SanDiego, CA, United States) at the Beijing Genomics Institute (Shenzhen, China). Genomic DNA was sheared randomly to construct three read libraries with length of 300bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, United States) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4000 system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software.

The whole-genome sequencing was implemented using an Illumina HiSeq 4,000 system (Illumina, SanDiego, CA, United States) at the Beijing Genomics Institute (Beijing, China). Genomic DNA was sheared randomly to construct three read libraries with length of 300 bp by a Bioruptor ultrasonicator (Diagenode, Denville, NJ, United States) and physico-chemical methods. The paired-end fragment libraries were sequenced according to the Illumina HiSeq 4,000 system’s protocol. Raw reads of low quality from paired-end sequencing (those with consecutive bases covered by fewer than five reads) were discarded. The sequenced reads were assembled using SOAPdenovo v1.05 software. Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values between these strains and other related strains were calculated using the Genome-to-Genome Distance Calculator (GGDC, version 3.0; http://ggdc.dsmz.de/ggdc.php; Auch et al., 2010 (link)) and with the ezbiocloud platform (Yoon et al., 2017b (link)), respectively.

Inbred lines of the mandarin fish species S. chuatsi, S. kneri, S. scherzeri and C. whiteheadi were obtained from the Chinese Perch Research Centre of Huazhong Agricultural University. Genomic DNA was extracted from the muscle of S. chuatsi (female), S. kneri (male), S. scherzeri (male), and C. whiteheadi (male) with a standard phenol-chloroform method. The diploid adult female S. chuatsi genome was sequenced on a PacBio RS II platform using P6-C4 chemistry (Pacific Biosciences, Menlo Park, CA, USA, 50× raw-read coverage). The transcriptome libraries of S. chuatsi brain, muscle, liver and gut tissues were prepared and sequenced using the Illumina HiSeq 4000 system (HiSeq 4000 SBS Kit, paired-end). The diploid adult male S. kneri, S. scherzeri and C. whiteheadi libraries for genome resequencing were prepared with the standard procedures for 300-bp paired-end libraries, and additional libraries were constructed using a Nextera^® Mate-Pair Library Preparation Kit (Illumina, San Diego, CA, USA). Libraries were sequenced on an Illumina HiSeq 4000 system. Library construction and sequencing were performed by the Beijing Genomics Institute (BGI, Wuhan, Hubei, China).

Total RNA was isolated from roots or shoots of 3-day old etiolated seedlings treated with air or 4 hours ethylene gas using TRIzol reagent (Invitrogen). For mRNA library construction, in briefly, the mRNA was isolated by NEBNext Poly(A) mRNA Magnetic Isolation Module and fragmented at 94°C for 15mins. Then the cDNA was synthesis by NEBNext Ultra Directional RNA Library Prep Kit for Illumina. The PCR reactions were conducted by using different index primers (NEBNext Multiplex Oligos for Illumina). The PCR products were purified by Agencourt AMPure XP Beads (Beckman Coulter). The quality of the libraries was assessed by Bioanalyzer (Agilent High Sensitivity Chip). The libraries then were sequenced on Hiseq 4000 Systems (Illumina).
For small RNA library construction, in briefly, the cDNAs were synthesized using NEBNext Small RNA Library Prep Set for Illumina. The PCR reaction was amplified by different Index primers and the PCR products were first purified by the Agencourt AMPure XP Beads, and then selected the size using 6% PolyAcrylamide Gel. The ~140 bp bands corresponding to miRNAs were isolated. The library quality was assessed on Bioanalyzer (Agilent High Sensitivity Chip). The libraries were sequenced on Hiseq 4000 Systems (Illumina) after assessed on Bioanalyzer.

For each sample, 120 ng of genomic DNA in 60 μL of 10 mM Tris-HCl, pH 8.0 buffer, was placed into 1.5 mL RNase-/DNase-free, low binding microcentrifuge tubes. Library creation steps were performed by the IIHG Genomics Division, and included DNA shearing using the Covaris Adaptive Focused Acoustics^TM process (Covaris E220 Focused-ultrasonicator; Covaris, Inc., Woburn, MA, United States), and DNA fragment purification and end polishing (KAPA Hyper prep kits; Kapa Biosystems, Inc., Wilmington, MA, United States) prior to ligation to indexed adaptors. The library size distribution was validated using the Agilent 2100 Bioanalyzer Instrument (Agilent Technologies, Santa Clara, CA, United States), and quantified using the q-PCR KAPA library amplification module following manufacturer instructions (Kapa Biosystems, Inc.). The indexed libraries were normalized, pooled, and clustered on a flow cell using the cBOT Cluster Generation System (Illumina, Inc., San Diego, CA, United States) and sequenced on the Illumina HiSeq 4000 System (Illumina, Inc.) in high output mode (1 lane, 2 × 150 bp). FASTQ files are accessible at ENA (Study Accession: PRJEB23134) and NCBI repositories (BioProject ID: PRJNA414922), and MG-RAST contains QA/QC and analyses of metagenomes (MG-RAST project mgp21252).