Gel extraction kit

RACE amplification was performed to obtain the full-length cDNA sequence of PsTOE3. The primers PsTOE3GSP5′ and PsTOE3GSP3′ were designed for RACE amplification based on the partial AP2 EST sequence from RNA-seq sequencing (Supplementary Data Table S1) [46 (link)]. cDNA synthesis and RACE PCR were performed using the SMART™ RACE cDNA amplification kit (ClonTech, USA) according to the manufacturer’s instructions. The objective fragments were collected using DNA and a gel extraction kit (Tiangen, Beijing, China), and then ligated into pMD18-T vector (TaKaRa, Dalian, China), and propagated in Escherichia coli DH5α. The positive colonies were sequenced by Sangon Co., Ltd. (Shanghai, China).

The PRMT5-minigene, a segment of PRMT5 including the region from exon 1 to exon 4, was amplified by polymerase chain reaction (PCR) from genomic DNA. The human HNRNPH1 and SRSF3 open reading frames (ORFs) were amplified by RT-PCR. The primers are listed in Supplementary Table S1. Next, the PCR products were gel-purified with the Gel Extraction Kit (Takara, Beijing, China) and then in-fusion cloned into the pcDNA 3.1-FLAG vector, which had been digested by BamHI and XhoI restriction endonucleases (NEB, Beijing, China) using the ExonArt Seamless Cloning and Assembly Kit (Exongen, Chengdu, China). The full-length PRMT5 (Myc-PRMT5L) and short-length PRMT5 ( Myc-PRMT5S, which was translated from PRMT5-ISO5) plasmids were generous gifts from Prof. Jiuyong Xie (Department of Physiology & Pathophysiology, University of Manitoba) as described [46 (link)].
The normal siRNA and In vivo siRNA (2′ O-methyl + 5′ cholesterol-modified) against HNRNPH1 or SRSF3 and nonsense siRNA (RiboBio, Guangzhou, China) were dissolved in rNase-free water for cell transfection or phosphate buffered saline (5 nmol siRNA with 50 μL PBS) for intratumoral injection. The sequences of si-HNRNPH1 and si-SRSF3 are shown in Supplementary Table S1 [47 (link),48 (link)].

Total RNA was extracted from floral buds using the EASYspin plant RNA Extraction Kit (Aidlab, China) following instructions from the manufacturer. First-strand cDNA was synthesized from 1 μg of the DNase I-treated RNA, using adaptor primers and M-MLV Reverse Transcriptase (TaKaRa, Japan). Initial amplification for core sequences were based on homologous cloning. The PCR reagents were composed of 1 μL cDNA, 0.5 μL of each primer (10 mM each), 2.5 μL Ex Taq buffer, 2 μL dNTP (2.5 mM each), 0.3 μL Ex Taq plymerase (TaKaRa, Japan) and adjusted with water to a final volume of 25 μL. PCR was performed with a 3 min 95°C denaturation step, followed by 35 cycles of 30 s at 95°C, 30 s annealing at 52–57°C, a 30–60 s extension at 72°C and a final extension period of 10 min. The PCR products were purified with the gel extraction kit (TaKaRa) and cloned into pMD18^®-T vector (TaKaRa). Ligation products were transformed into Escherichia coli Top10 cells (Aidlab China) following instructions by the manufacturer. Then we used 3’ RACE and 5’ RACE system kits (TaKaRa) to obtain the 3’- and 5’-end sequences of each gene. Full-length cDNA of each gene was obtained by PCR-based cloning with gene-specific forward and reverse primers designed according to the corresponding 3’- and 5’-end sequences. Names and sequences of the primers used in this study are presented in Tables 1 and 2.

Bisulfite sequencing was performed using specific tissue samples and cells. The primers used for bisulfite sequencing of the KvDMR1 fragment are listed in Supplemental Table 3. The amplified area contained 28 CpG sites. Amplified PCR products were purified using a gel extraction kit (Takara) and ligated into the pMD19-T plasmid using the TA-cloning system (Takara). The sequences were analyzed using 3730 DNA Analyzer polymers (Applied Biosystems, Carlsbad, CA).

Escherichia coli DH5α was used as an intermediate host to construct plasmids. Pichia pastoris GS115 was used as a recipient strain for gene expression. Microorganisms used for the evaluation of the antifungal activity of compound J075-4187 of the ChemDiv compound library were: Aspergillus nidulans (ATCC 10074), Fusarium graminearum (ATCC 56091), Aspergillus flavus (ATCC 204304), Botrytis cinerea (ATCC48340), Fusarium oxysporum sp. cucumebrium Owen (ATCC42357), and Saccharomyces cerevisiae (ATCC 4226). Test fungal strains were cultured in Potato Dextrose agar. E. coli DH5α was grown at 37°C in an LB medium. P. pastoris GS115 was grown at 30°C overnight at 280 rpm in YPD medium. The pPICZ A plasmid was used to construct an expression vector. The medium for the E. coli cells containing the plasmid was supplemented with ampicillin (100 μg/ml). EcoRI, NotI, and a gel extraction kit were obtained from TaKaRa (Japan). 4-Nitroacetanilide was purchased from Beijing Bailingwei Technology Co., Ltd., China. An EasySelectTM Pichia Expression Kit was obtained from Invitrogen Co. (USA). The top-ranked 30 compounds derived from the ChemDiv database by structure-based pharmacophore virtual screening were purchased from ChemDiv, Inc. (USA). Unless otherwise noted, analytical-grade reagents and solvents were obtained from commercial sources.

Primer pairs designed based on the alignment results of the NS3 region of all BovHepV available in GenBank database were used to detect the presence of BovHepV RNA in blood-sucking ticks. Using nested RT-PCR with the primer pair BovHepV-F1/BovHepV-R1 and BovHepV-F2/BovHepV-R2, the PCR product with 493 bp was confirmed by Sanger sequencing. In addition, over-lapping primers were designed to obtain the complete genome sequence of the BovHepV identified in this study. All primers used in this study were listed in Table 1.
The PCR products of the expected size, according to each set of primers, were purified using Gel Extraction kit (TaKaRa, Dalian, China) after electrophoresis. The purified DNA was cloned into pMD19-T vector (TaKaRa, China), and the resulting plasmid was used to transform competent E. coli cells. Positive inserts were confirmed by PCR, and further sequenced by Sangon Biotechnology Company (Shanghai, China). To prevent contamination, the preparation of the PCR mix and the addition of the template DNA were performed in separate rooms using dedicated pipets and filtered tips.

A DNeasy tissue kit (Qiagen) was used to isolate genomic DNA from K. pneumoniae KCTC2242. Restriction enzymes and T4 DNA ligase were purchased from Takara Shuzo. An Axy PrepTM kit (Axygen) was used to isolate bacterial plasmid DNA. A gel extraction kit (Takara, A550) was used for large-scale isolation of plasmid DNA from gels.