Eagent (Molecular Investigation Center) from 10-day-old seedlings from LUCL met1-3, LUCL ago4-6 and LUCL drm2-6 plants as previously described [46]. For the RT-PCR in Figures 3 and 4, older leaf tissue from LUCL met1-3, LUCL ago4-6, and LUCL drm2-6 plants was utilized. For the RT-PCR in Figure 5, 10-day-old, chemical-treated seedlings were applied. cDNA was synthesized from 5 g (14 g for Figure 5) of DNaseI (Invitrogen)-treated total RNA employing reverse transcriptase (Fermentas) and oligo-dT (Fermentas) as previouslyDinh et al. Silence 2013, four:1 http://www.silencejournal/content/4/1/Page ten ofdescribed [21]. The sequences of primers are listed in Further file 1: Table S1.Chemical screeningSmall molecule compounds employed for the chemical screen consist of: 1,200 from LifeSciences, two,000 from Spectrum and 400 from Myria/Sigma in the UCR small compounds collection [47]; 4,204 from a triazine-tagged library [48,49]; two,768 from Clickables [50] and 3,580 from LATCA [51]. The screening was performed in the Chemical Screening Facilities at UC Riverside.Added fileAdditional file 1: Figure S1. LUCL is not regulated by the miRNA pathway. Luciferase luminescence of LUCL, LUCH, and seedlings from a number of F2 populations (#101, 103 and 104) of dcl1-7 crossed to LUCL. Inside the F2 population, none on the seedlings showed de-repression of luciferase activity. Table S1. DNA oligonucleotides utilized in this study. Table S2. Conversion prices for the bisulfite sequencing experiments. Abbreviations 5-aza-2-dC: 5-aza-2-deoxycytidine; bp: base pair; DHF: dihydrofolate; DHFR: dihydrofolate reductase; DMSO: Dimethyl sulfoxide; GTP: guanosine triphosphate; miRNA: microRNA; MTX: methotrexate (also referred to as amethopterin); nt: nucleotide; PCR: polymerase chain reaction; RdDM: RNA-directed DNA methylation; RT-PCR: reverse transcription-PCR; SAH: S-adenosylhomocysteine; SAM: S-adenosyl methionine; siRNA: tiny interfering RNA; ssRNA: single-stranded RNA; TAIL-PCR: thermal asymmetric interlaced PCR; TGS: transcriptional gene silencing; THF: tetrahydrofolate. Competing interests The authors declare that they’ve no competing interests. Authors’ contributions TTD and XC wrote the manuscript. TTD created the chemical screen and performed genetic analyses of LUCL, Southern blot analyses, bisulfite sequencing analyses, luciferase imaging, RT-PCR and McrBC analysis. TTD and MOL performed the chemical screen. MOL performed the luciferase imaging for the MTX-treated seedlings. SYW did the 5-aza-2-dC treatment and RT-PCR of treated seedlings.CITCO XL, TTD and LA generated the LUCL met1-3, LUCL ago4-6 and LUCL drm2-6 genotypes and analyzed them.Sofosbuvir SL performed the McrBC evaluation on LUCH and LUCL.PMID:24982871 AD and SRC provided chemicals, performed modest molecule perturbations and provided intellectual help with regards towards the chemical screen. BZ transformed LUCL into rdr6-11. XC constructed the reporter plasmid, conceived and guided the project. All authors study and authorized the final manuscript. Acknowledgments The perform was supported by a grant from the National Science Foundation (MCB-1021465) and by Howard Hughes Health-related Institute and Gordon and Betty Moore Foundation (by way of Grant GBMF3046) to XC. TTD was supported by a National Science Foundation ChemGen IGERT plan (DGE0504249). Author specifics 1 Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, CA 92521, USA. 2NSF ChemGen IGERT system, University of California, Riversid.