Hiseqtm 4000 platform

To obtain enough RNA, from PQs, PKs, WMs and WFs samples using TRIzol reagent and checked the total RNA quality assessed an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Three replicates of each caste WMs, WFs, PKs and PQs, were pooled to obtain enough RNA and build cDNA libraries using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB). In brief, using Oligo(dT) beads to extract the total RNA, enriched from mRNA and fragmentation buffer were used to transcribed into short fragments into cDNA with random primers. Second-strand cDNA was synthesized by using DNA polymerase I, RNase H, dNTPs and buffer. The cDNA fragments were then purified with the QiaQuick PCR extraction kit, poly(A) end-repaired was tailed, and attached to the Illumina sequencing adapters. Ligation products were selected in size by agarose gel electrophoresis, PCR amplified and sequenced using the Illumina HiSeqTM 4000 platform by Gene Denovo Biotechnology Co. (Guangzhou, China)^{66 (link),67} .

Fresh leaves of 11 Stenophora species/subspecies, i.e., D. banzhuana, D. biformifolia, D. collettii, D. deltoidea, D. futschauensis, D. gracillima, D. nipponica, D. nipponica subsp. rosthorni, D. spongiosa, D. tokoro, D. zingiberensis, were field-collected and dried with silica-gel. The voucher specimens were deposited at Herbarium of Institute of Botany, Jiangsu Province and Chinese Academy of Sciences (NAS) [details about sampling information can be found in Gao et al. (2008) (link)]. For each species/subspecies, genomic DNA was extracted from silica gel-dried leaves using DNAsecure Plant Kit (Tiangen Biotech, Beijing, China), following the manufacturer’s protocol. DNA concentration and integrity were measured by Agilent 2100 BioAnalyzer and agarose gel electrophoresis. Paired-end library with insert size of 350 bp was constructed for each species/subspecies by using the Genomic DNA Sample Prep, and then sequenced on the Illumina HiSeqTM 4000 platform (Illumina, San Diego, California, USA) according to the paired-end 2 × 150 bp protocol. Library construction, genome sequencing and raw data filtering were conducted by Novogene Bioinformatics Technology Co., Ltd., Beijing, China.

According to the manufacturer’s instructions, the RNAsimple Total RNA Kit (Tiangen, Beijing, China) was used to extract total RNA from the RT, LF, SC and IN of three batches of A. orientale. The quality and quantity of RNA were examined by spectrophotometric analysis (BioDrop spectrophotometer, BioDrop Technologies, Cambridge, UK) and agarose gel electrophoresis. After total RNA was extracted, the mRNA was enriched with magnetic beads, cut into short fragments, and then reverse transcribed into first-strand cDNA. The second-strand cDNA were synthesized by RNase H, DNA polymerase I and dNTPs. Next, the cDNA fragments was purified and ligated to Illumina sequencing adapters. The ligation products were amplified by polymerase chain reaction, followed by high-throughput sequencing on the Illumina HiSeqTM 4000 platform (Gene Denovo Biotechnology Co. Ltd, Guangzhou, China).

Total RNA of the six biological replicas (Am7T-1, Am7T-2, Am7T-3, Am10T-1, Am10T-2, and Am10T-3) from N. ceranae-treated groups and six biological replicas (Am7CK-1, Am7CK-2, Am7CK-3, Am10CK-1, Am10CK -2, and Am10CK-3) from control groups were respectively extracted using Trizol (Life Technologies) following the manufacturer’s instructions, and checked via 1% agarose gel eletrophoresis. Subsequently, rRNAs were removed to retain mRNAs and ncRNAs, which were fragmented into short fragments by using fragmentation buffer (Illumina) and reverse transcripted into cDNA with random primers. Next, second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP (dUTP instead of dTTP), and buffer. The cDNA fragments were purified using QiaQuick PCR extraction kit (QIAGEN), end repaired, poly (A) added, and ligated to Illumina sequencing adapters. UNG (Uracil-N-Glycosylase) (Illumina) was then used to digest the second-strand cDNA. Ultimately, the digested products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced on Illumina HiSeq^TM 4000 platform (Illumina) by Gene Denovo Biotechnology Co. (Guangzhou). All RNA sequencing data produced in our study are available in NCBI Short Read Archive database (http://www.ncbi.nlm.nih.gov/sra/) and could be available on Sequence Read Archive (SRA) database and connected to BioProject PRJNA406998.

SK-Hep1 cells in triplicate were treated with simvastatin (20 μM) and control for 24 h. Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and checked using RNase free agarose gel electrophoresis. mRNAs were isolated and fragmented to about 200 base pairs length and reverse transcribed into cDNA using the QuantiTect Reverse Transcription kit (Qiagen). The cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, Netherland), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The cDNA library products were size selected by agarose gel electrophoresis, PCR amplified and sequenced using Illumina HiSeqTM4000 platform (Illumina, San Diego, CA). Transcript-level expression analysis of sequencing data was performed using HISAT and StringTie software (http://ccb.jhu.edu/software,shtml) [24 (link)]. Differential transcripts of chemokines with stringent cutoff coefficient of less than 0.05 were obtained to align to GO database (http://www.geneontology.org) for protein functional annotation corresponding to immune population.

The frozen tissues were separately ground using liquid nitrogen in a mortar and pestle. The cDNA libraries were constructed by following the previous methodology [16 (link)]. In brief, total RNA from each sample was separately extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA). The quality of the extracted total RNA was evaluated by electrophoresis (1% agarose gel).
The mRNA molecules were purified using an Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany), which were used as templates to synthesize the first-strand cDNA using random hexamer primers, and ultimately to synthesize second-strand cDNA libraries. In this study, twelve cDNA libraries including three males mid-guts, three females mid-guts, three males testes, and three females ovaries were prepared to construct four cDNA libraries, each with three independent biological replicates that were separately prepared by sequencing through an Illumina HiSeq^TM 4000 platform at MicroAnaly Gene Technologies Co., Ltd. (Wuhan, Hubei, China). These data have been made available at the NCBI Sequence Read Archive (SRA BioProject Acc. No. PRJNA647692).