Sequel sequencing kit 2

The RNA extracted from five tissue types was mixed into one specimen to establish SMRT library. Full-length cDNA was produced using a SMARTer PCR cDNA Synthesis Kit (Clontech), and isoform sequencing (Iso-Seq) libraries were constructed using a SMRTbell™ Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA, USA). Sequencing was performed on a PacBio Sequel II instrument with a Sequel™ Sequencing Kit 2.0 (Pacific Biosciences). Functional annotations were conducted using BLAST (version 2.2.26) against different protein and nucleotide databases including the NR database, Swissprot database, Gene Ontology (GO) database, eggNOG (Evolutionary Genealogy of Genes: Non-super-vised Orthologous Groups) database, and KEGG (Kyoto Encyclopedia of Genes and Genomics) database. Principal component analysis (PCA) is an important analytical method that analyzes the multiple sets of data and interprets it with fewer principal components, while visualizing differences and interpreting most characteristics of the original data (Wang et al., 2012 (link)). Heatmap was plotted by an online platform for data analysis and visualization (https://www.bioinformatics.com.cn).

For Pacbio-based RNA sequencing (RNA-seq), the samples for Pac-Bio library construction were the mixed RNAs from all five plant samples. The full-length cDNAs were synthesised using a SMARTer PCR cDNA Synthesis Kit (Clontech, USA). The cDNA library was constructed using the SMRTbell™ Template Prep Kit 1.0, following the manufacturer’s instructions. The cDNA library was sequenced using a Sequel™ Sequencing Kit 2.0 with a PacBio Sequel system (Pacific Biosciences, USA). The raw sequence data were deposited in the SRA database and are available under accession number SRR10352319.

We utilized TRIzol reagent (Invitrogen, Waltham, MA, USA) to extract total cellular RNA in line with specific protocols. After purification, we adopted DNase I (Qiagen, Hilden, Germany) for enzymatic digestion. Later, we utilized the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) to assess RNA purity and content. Meanwhile, we adopted SMARTer PCR cDNA Synthesis Kit (Takara Biotechnology, Shiga, Japan) to generate cDNA and constructed libraries with SMRTbell™ Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA, USA). Subsequently, we adopted Sequel™ Sequencing Kit 2.0 (Pacific Biosciences) for library sequencing (Shanghai Personalbio Technology Co., Ltd., Shanghai, China).

In PacBio sequencing, the total RNA of E4.5 gonads was reverse-transcribed to cDNA using SMARTer PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA). After PCR amplification, BluePippin was used to select long-length fragments. Library construction was conducted using SMRTbell™ Template Prep Kit 1.0 (Pacific Biosciences, Menlo Park, CA, USA), and then was sequenced using Sequel™ Sequencing Kit 2.0. During Illumina sequencing, mRNA was separately purified from the total RNA of E4.5, E5.5, and E6.5 gonads using poly-T oligo-attached magnetic beads. cDNA was synthesized using random oligonucleotides and Super Script II for the first strand, and DNA Polymerase I and RNase H for the second strand. After the adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated. Fragments were purified using the AMPure XP system (Beckman Coulter, Brea, CA, USA) for selecting 400–500 bp of cDNA, then PCR-amplified using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction and quantified using the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). The sequencing library was sequenced on Illumina NovaSeq 6000 platform. All transcriptome sequencing was performed by Shanghai Personal Biotechnology Cp. Ltd. (Shanghai, China).

For both PacBio and Illumina DNA sequencing, all steps were carried out by Novogene (Hong Kong) as part of the Global Ant Genomics Alliance (GAGA, Boomsma et al. 2017 ). DNA from a single haploid male was extracted using a Sodium Dodecyl Sulfate (SDS) protocol following Pippel et al. (2020) (link) and a SMRTbell library was prepared using the SMRT bell Template Prep Kit 1.0-SPv3 (Pacbio, 100-991-900). DNA quantification was performed using a Qubit fluorometer (Thermo Fisher) and purity was assessed with an agarose gel electrophoresis. The extraction from a single male yielded 9.89 μg of DNA, at a concentration of 86 ng/μL (A_260/280 = 1.76, A_260/230 = 1.20). DNA fragmentation was assessed through an Advanced Analytical Fragment Analyzer (AATI, mean size: 18 317 bp) prior to size selection (BluePippin, Sage Sciences, cutoff: 10 kb). The sample was loaded onto 4 SMRT cells with the Sequel Sequencing Kit 2.0 following PacBio recommendations and sequenced on a PacBio Sequel platform.

The pretarget captured sequencing libraries with Illumina format adapters were pooled and subjected to single‐molecule real‐time (SMRT) sequencing template construction using a SMRTbell Template Prep Kit 1.0—SPv3 (Pacific Biosciences). The posttarget captured Illumina sequencing libraries were similarly pooled and further subjected to SMRT sequencing library construction. The amplicon template preparation and sequencing protocol was used, with minor modifications: DNA was purified with 1.8× AMPure PB beads, and library size was estimated using a TapeStation instrument (Agilent). Sequencing primer annealing and polymerase binding conditions were calculated with the SMRT Link v5.1.0 software (Pacific Biosciences). Briefly, sequencing primer v3 was annealed to the sequencing template, and then polymerase was bound to templates using a Sequel Binding and Internal Control Kit 2.1 (Pacific Biosciences). Sequencing was performed on a Sequel SMRT Cell 1M v2. Sequencing movies were collected on the Sequel system for 10 hours with a Sequel Sequencing Kit 2.1 (Pacific Biosciences).