2.2 Sample preparation, library construction and sequencing
An isogenic colony was started from a single parthenogenetic female ofM. dirhodum and was maintained alone on wheat seedlings prior to the collection of insects for sequencing. Two hundred milligrams of fresh mixed M. dirhodum(including first- to fourth-instar nymphs and winged and wingless adults) were collected for DNA extraction and genome sequencing. Total genomic DNA was extracted using a Blood & Cell Culture DNA Mini Kit according to the manufacturer’s protocol (Qiagen, Hilden, Germany). For short-read sequencing, a paired-end library (2×150 bp) with short insert sizes of approximately 500 bp was constructed using the VAHTSTM Universal DNA Library Prep Kit for Illumina V2 (Vazyme, Nanning, China) and then sequenced on an Illumina NovaSeq 6000 platform (San Diego, CA, USA). For long-read genomic sequencing, the PacBio SMRTbell 15 kb library was constructed using a SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, CA, USA) and then sequenced on the PacBio Sequel II platform for circular consensus sequencing (CCS) (Pacific Biosciences, CA, USA).
To assist chromosome-level assembly, the Hi-C (high-throughput chromosome conformation capture) technique was applied to capture genome-wide chromatin interactions. Approximately 200 mg of fresh mixedM. dirhodum (including first- to fourth-instar nymphs and winged and wingless adults) was ground in 2% formaldehyde to allow cross-linking of cellular protein, and approximately 100 μg of DNA was extracted. Subsequently, chromosome integrity and cross-linked protein residues were assessed. Chromatin digestion was performed with the restriction enzyme Mbo I. Biotinylated residues were added during repair of the sticky ends, and the resulting blunt-end fragments were ligated under dilute conditions (Lieberman-Aiden et al., 2009; Belton et al., 2012; Rao et al., 2014; Belaghzal et al., 2017; Pan et al., 2021). The DNA was extracted and randomly sheared to fragments of 300–500 bp. The biotin-labeled fragments were isolated with magnetic beads. The next four steps, including end repair, dA tailing, adapter ligation and DNA purification, were accomplished by adding the corresponding reaction components sequentially. The library quantity was estimated by Qubit 2.0, an Agilent 2100 instrument (Agilent Technologies, Santa Clara, CA, USA), and quantitative PCR. The Hi-C library was then sequenced using the Illumina NovaSeq 6000 platform with paired-end 150 bp reads.
For PacBio full-length transcriptome sequencing, total RNA was isolated from fresh mixedM. dirhodum (including first- to fourth-instar nymphs and winged and wingless adults of equal quality) using an EASYspin Plus Cell/Tissue RNA Isolation Kit (Aidlab Biotechnologies, Beijing, China) and quantified using a NanoDrop ND-2000 spectrophotometer (NanoDrop products, Wilmington, DE, USA). Ten micrograms of total RNA wee reverse transcribed into cDNA using a SMARTer PCR cDNA Synthesis Kit (Takara, Dalian, China) following the manufacturer’s protocols. The SMRT library was constructed using the SMRTbell template prep kit (Takara) following the manufacturer’s protocols. The library was sequenced on the PacBio Sequel II platform, and SMRTlink was used to obtain full-length consensus isoform sequences.
For Illumina transcriptome sequencing, total RNA was isolated from winged or wingless M. dirhodum of third- and fourth-instar nymphs and adults of equal quality using an EASYspin Plus Cell/Tissue RNA Isolation Kit (Aidlab Biotechnologies, Beijing, China) and then quantified using a NanoDrop ND-2000 spectrophotometer. cDNA libraries were constructed using a VAHTSTM mRNA-seq V3 Library Prep Kit (Vazyme, Nanjing, China). A total of 18 libraries were constructed with three biological replicates per sample. Sequencing was performed on an Illumina NovaSeq instrument (Illumina, San Diego, CA, USA), and 150 bp paired-end reads were generated.