Oligo dt magnetic beads

Total RNA of the four accessions was extracted using Trizol reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The RNA concentrations were determined using a Qubit® RNA Assay Kit and a Qubit®2.0 Fluorometer (Life Technologies, CA, USA), and the RNA integrity was assessed using an RNA Nano 6000 Assay Kit with an Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Poly(A)-containing mRNAs were purified from the total RNA with oligo (dT) magnetic beads (Illumina, San Diego, CA). Then, mRNA random fragmentation, cDNA synthesis, purification and PCR amplification were performed according to the Illumina RNA-Seq protocol. The four cDNA libraries were sequenced with a read length of 194 bp on a HiSeq 2000 system with a paired-end module at Biomarker Co. Ltd. (Beijing, China).

To obtain poly (A) mRNA from the total RNA, oligo (dT) magnetic beads were used (Illumina). The RNA was broken down into short fragments by the addition of fragmentation buffer. These short fragments were then used as templates for first-strand cDNA synthesis with random hexamers and reverse transcriptase (Illumina). To synthesize the second strand of the cDNA, a solution of RNase H (Illumina), DNA polymerase I (Illumina), dNTPs, and buffer was used. The ends of the resulting double-stranded cDNA fragments were repaired, and adapters were ligated. The final version of the cDNA library was prepared from these products by purification and subsequent amplification by PCR. Using the Illumina Hiseq 2000 platform, the four prepared cDNA libraries were sequenced, resulting in 100-bp paired-end reads.

Plants from the inbred maize line CAL were grown in a greenhouse with a 16 h/8 h light/dark cycle at 20–25°C. Immature embryos were collected from the maize ears 10–12 days after pollination. The immature embryos (1.0–1.2 mm) were isolated and placed on N6 medium with 1.5 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and subjected to aphotic culturing at 27°C. After culturing for 0, 1, 2, 4, 6, and 8 days (D0, D1, D2, D4, D6, and D8) at 27°C, the immature embryos were collected and stored at −70°C for RNA extraction. Meanwhile, immature embryos cultured without 2,4-D (N1, N2, N4, N6, and N8) were also collected as control. More than 50 immature embryos were collected for each sample, and three biological replicates of each sample were used for the following RNA-Seq.
The extracted RNA was inserted into a 1% agarose gel to assess the RNA integrity. RNA yield and purity were checked using a Nano-drop ND-1000. mRNAs were isolated from total RNA using oligo(dT) magnetic beads (Illumina, San Diego, CA, USA). RNA fragmentation, cDNA synthesis, and PCR amplification were performed according to the Illumina RNA-Seq protocol. cDNA libraries were sequenced with a read length of 150 bp (paired-end) using an Illumina HiSeq 2500 System at Berry Genomics (Beijing, China).

Total RNA was extracted from individual control, wounded and defoliated plants at 2, 6, and 24 h using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). After precipitation, the RNA was purified using Qiagen’s RNeasy Kit with on-column DNase I digestion according to the manufacturer’s instructions. RNA concentrations were measured using the Bioanalyzer 2100 (Agilent Technologies, Inc., Waldbronn, Germany), and the RNA integrity was analyzed on a 1.0% (w/v) agarose gel. Approximately 5 to 8 µg total RNA was used to construct each RNA-seq library. Poly (A) mRNAs were isolated from the total RNA using oligo (dT) magnetic beads (Illumina, San Diego, CA). RNA fragmentation, cDNA synthesis, and PCR amplification were performed according to the Illumina RNA-seq protocol (Cat # RS-100-0801). Seven cDNA libraries, which were isolated from mechanically wounded and defoliated material at 2, 6, and 24 h and the control, were sequenced using the Illumina HiSeq 2000 System at the Chinese National Human Genome Center (Shanghai, China). The sequence reads generated in this study have been deposited in the NCBI sequence read archive (SRA065691).

Total RNA from control and induced calli was purified using oligo(dT) magnetic beads and fragmented into small pieces according to the manufacturer’s instruction (Illumina, San Diego, CA USA). The cleaved fragments were then used to synthesize first-strand cDNA with random hexamer primiers and reverse transcriptase (Invitrogen, Carlsbad, CA USA). Second-strand cDNA was synthesized using DNA polymerase I (Invitrogen, Carlsbad, CA USA), dNTPs and RNase H. Following, the cDNA fragment were amplified by PCR and purified after removal of the ligation adaptors and end-repair to construct the final library. Three libraries with the insert size ranging from 150 to 250 bp were sequenced on the Illumina HiSeq-2000 platform using the 100 bp paired-end approach. In total, 74,881,134, 80,382,270 and 77,355,892 raw reads were generated by Solexa/Illumina sequencing in three libraries respectively, which had been deposited in Sequence Read Archive database in NCBI, Accession No. SRA319923.

The dissected venom gland tissues were submerged and homogenised in a 1 ml glass homogeniser with TRIzol solution (Invitrogen, Carlsbad, CA, USA) under sterile conditions. This was followed by the addition of 20% chloroform, centrifugation and RNA-free DNAase I treatment to separate RNA from cellular debris and residual DNA. The isolated RNA was then pelleted with isopropyl alcohol and washed with 75% ethanol. Polyadenylated mRNA (poly(A)⁺ mRNA) was subsequently purified from 20 mg of total RNA using oligo (dT) magnetic beads as per the (Illumina, San Diego, CA, USA) manufacturer’s instructions. Two rounds of poly(A)⁺ mRNA isolation were performed.

WT and overexpression strains cultured for 6 days were used for RNA-seq. Total RNA was extracted using the Trizol reagent kit (Invitrogen) according to the manufacturer’s protocol, and RNA quality was assessed. Qualified RNA (RIN value > 7) was purified using oligo (dT) magnetic beads (Illumina) and used to synthesize cDNA. A sequencing library was constructed using synthetic cDNA and sequenced on the Illumina Novaseq 6000 platform at Gene Denovo Biotechnology Co., Ltd (Guangzhou, China). For data analysis, raw data were filtered for data quality assurance. The clean data were mapped to the reference genome (NCBI: GCA_000150955.2 ASM15095v2, P. tricornutum). Gene abundance was quantified using StringTie v1.3.1 and RSEM software. The DESeq2 software was used to detect differentially expressed genes (DEGs) between the WT and overexpression lines. Genes with a false discovery rate (FDR) < 0.05 and |log₂fold change | ≥ 1 were considered as DEGs^{58 (link)}.