Qiaquick gel extraction

For minigene assays, Hep3B or SH-SY5Y cells were seeded in six-well plates at a density of 4 × 10⁵ or 8 × 10⁵, respectively, in 2 mL MEM or DMEM 5% FBS and grown overnight. Cells were transfected using Lipofectamine 2000 (Invitrogen) with 2 μg per well of the corresponding plasmid minigene. For SSO treatment, cells were cotransfected with 2 μg of wild type or mutant minigenes and 50 nM of each SSO. Cells were harvested by trypsinization 48 h after transfection. Total RNA was isolated using TRIzol Reagent (ThermoFisher) and phenol–chloroform extraction. cDNA synthesis was performed using NZY First-Strand cDNA Synthesis Kit (NZYtech).
Splicing analysis was carried out by polymerase chain reaction (PCR) amplification with FastStart Taq DNA Polymerase (Roche) using specific primers SD6 (5′- TCTGAGTCACCTGGACAACC-3′) and SA2 (5′- ATCTCAGTGGTATTTGTGAGC-3′) for pSPL3 minigenes and PL3 (5′-GGGAGACCCAAGCTGGCTA-3′) and PL4b (5′-AGTCGAGGCTGATCAGCGG-3′) for the pCR3.1 minigene. The amplification products were analyzed by 2% agarose gel electrophoresis. Pseudoexon bands were excised from the gel and purified using QIAquick Gel Extraction (Qiagen). The identity of each pseudoexon was confirmed by subcloning in pGEMT (Promega) and subsequent Sanger sequencing.

The molecular reagents TRIzolReagent, Oligo(dT)_12-18, RNase free DNAase I, RNase OUT, RNase H, Super Script III, Lipofectamine 2000 and High DNA Mass Ladder were purchased from Invitrogen (San Diego CA, USA). RNeasy Plus Mini Kit, QIA quick PCR Purification, QIA quick gel extraction and QIAprep Spin Mini prep Kit from Qiagen (Valencia CA, USA). The Pfu DNA polymerase was from Promega (Madison WI, USA). T4 DNA ligase was from Thermo Scientific (Waltham, MA, USA). EZNA Endo-free Plasmid DNA mini kit from OMEGA Biotek (Norcross GA USA) was used to purify the plasmid for transfection. The vector pUC18 was from Stratagene, (Agilent Technologies Company, Santa Clara, CA, USA) and pIRES-GFP from BD Biosciences Clontech (San Jose, CA, USA). GelRed Nucleic Acid Stain was from Biotium, Hayward CA, USA) HEK293 cells were obtained from ATCC (American Type cell Collection Manassas, VA, USA). Dulbecco’s modified Eagle medium (DMEM-F12) and fetal bovine serum were from GIBCO (Invitrogen Corporation, San Diego CA, USA). Adobe Photoshop was from ADOBE Systems Mountain View, CA, USA). All other reagents were purchased from Sigma-Aldrich (St. Louis MO; USA).

The homolog of Arabidopsis DMR6 (AtDMR6) in sweet basil (Ocimum basilicum), ObDMR6, was identified by TBLASTX search against the non-redundant transcriptomic sequence assembly generated from an O. basilicum cultivar, Dolly (Enza Zaden) (S1 Appendix), using the protein encoding sequence of AtDMR6 (GenBank accession: NM_122361) as a query. Three significant hits were identified based on query coverage and E-value. To amplify ObDMR6 from sweet basil cultivar Genoveser by PCR, we designed a pair of primers (ObDMR6-F: 5’-ATGGAAACGAAGGTCATTAGTG-3’; ObDMR6-R: 5’-CTAATTCTTGAATAGTTCCAGGCAG-3’) targeting the start and stop regions of the protein-encoding sequences of the identified transcripts from Dolly. Two PCR assays were preformed using Genoveser genomic DNA (gDNA) and complementary DNA (cDNA) as the templates and Phusion High-Fidelity DNA Polymerase (NEB) under the PCR conditions: initial denaturation at 98°C for 30 s; 35 cycles of 98°C for 30 s, 62°C for 15 s, and 72°C for 1.5 min; and a final extension at 72°C for 10 min. The resultant amplicons were separated by agarose gel electrophoresis, purified using QIAquick Gel Extraction (QIAGEN), cloned into pCR4Blunt-TOPO using Zero Blunt™ TOPO™ PCR Cloning Kit (ThermoFisher), and then subjected to Sanger sequencing. Sequence alignments were generated using CLUSTALX 2.1 [34 (link)].

RNA was extracted from 1 ml plasma aliquots with QIAamp UltraSens Virus RNA kit (Qiagen, Valencia, CA) and reverse-transcribed using SuperScript III First-strand Supermix and random hexamer primer (Invitrogen, Carlsbad, CA). DNA was extracted from LN cell suspensions with QIAamp Blood and Tissue kit (Qiagen). PCR amplification was carried out to amplify the pol gene encoding the first ∼250 amino acids of RT with primers as previously described [15] (link). PCR products were purified with QIAquick gel extraction or QIAquick PCR purification kits (Qiagen). Clones were prepared by Zero-Blunt TOPO cloning (Invitrogen) and transformation of Top10 E. coli (Invitrogen). Individual bacterial colonies were grown in LB-broth (Invitrogen), and DNA was extracted with QIAprep Spin Miniprep kits (Qiagen). The presence of the RT gene was verified by restriction digestion and visualization on a 1% agarose gel, and positive clones were sent for sequencing (GeneWiz, South Plainfield, NJ) with primers specific to HIV_HxB2 RT. Gene sequences were analyzed by Lasergene 10 (DNAStar, Madison, WI). Mutations were classified as NNRTI DRMs as defined by the Stanford University HIV Drug Resistance Database (http://hivdb.stanford.edu/) [30] (link).

Genomic DNA was obtained from R. pumilio ear biopsies. Genomic DNA PCR was performed using Q5 High-Fidelity DNA polymerase (NEB) according to the manufacturer's instructions. The PCR products were run on a 1.5% agarose gel, gel extraction using QIAQuick Gel extraction (Qiagen) was performed on any bands of the expected/appropriate size and fragments were sequenced using Sanger sequencing.