Hisequation 2500 platform

We sequenced the liver tissue transcriptomes to access a large number of diverse transcripts because the liver is a highly complex organ with a complex transcriptome (Shackel et al. 2002 (link), 2006 (link)). Total RNA was isolated using TRIzol reagent (Life Technologies Corp., Carlsbad, CA) according to the manufacturer’s protocols, and cleaned up using the RNeasy mini kit (Qiagen, Valencia, CA). RNA samples were quantified with the 2100 Bioanalyzer (Agilent Technologies). mRNA was enriched using beads with oligo (dT) and fragmented using fragmentation buffer. The first cDNA chain was synthesized using random hexamers, and the second chain was synthesized with buffer, dNTPs, RNase H, and DNA polymerase I. After the cDNA was purified using Agencourt AMPure XP (Beckman Coulter Inc., Atlanta, GA), the samples were blunt-end repaired and ligated with poly(A) and adapter sequences. Then, sizes were selected using Agencourt AMPure XP (Beckman Coulter Inc., Atlanta, GA). Each of the libraries was amplified by PCR. The libraries were quantified by RT-PCR, and transcriptome sequencing was performed on an Illumina HiSequation 2500 platform. Short sequence reads of paired end (PE) 100 bp were generated. All of the data were deposited into the NCBI Sequence Read Archive database (SRA run accession numbers are provided in Table S1).

For the methylome analysis, gDNA was extracted from the heads and thoraxes of four male and four female H. armigera, and pooled for sequencing. Insects were collected as adults from Bt cotton fields in Qiuxian (Hebei province, China, 36.81°N, 115.16°E) and reared for one generation in the insectaries at Rothamsted Research. Adults were snap-frozen in liquid nitrogen and gDNA extracted using the DNeasy Blood and Tissue Kit (QIAGEN). Methyl-MaxiSeq (Zymo Research) libraries were prepared from 100 ng of bisulfite-treated gDNA (EZ DNA Methylation-Lightning Kit). Bisulfite-converted DNA was amplified with a primer that contained part of an adaptor sequence plus four random nucleotides, followed by two additional amplifications to add on the remaining adaptor sequence and to barcode the fragments. PCR products were purified using the DNA Clean & Concentrator-5 (Zymo Research). Sequencing was run on the Illumina HiSequation 2500 platform. Sequence reads were aligned to the H. armigera genome using the bisulfite sequencing aligner software Bismark (Krueger and Andrews 2011 (link)).