17. Higher hybridization and wash temperatures may be required
when using Klenow probes because of their lower specificity
(Fig. 5). Caution: Ensure that all buffer components are fully
dissolved by preheating the solution to 35
C.
Acknowledgments
M.B., A.G., and D.P. developed the bioinformatics pipeline for TE
annotation, genome browser display, and northern blot probe
design. M.B., B.R., and C.H. performed the molecular genetic
and benchtop experiments. R.S., D. L.-C., A.C., and
J.V. provided B. distachyon mutant germplasm identified using
high-throughput sequencing and bioinformatics; work at the US
DOE Joint Genome Institute is supported by the Office of Science
of the US Department of Energy under Contract no. DE-AC0205CH1123 and by user agreement FP00004794 between JGI and
the USDA Agricultural Research Service. M.B., B.R., and
T.B. assembled the figures and wrote the manuscript. The authors
thank Hugues Renault for advice on culturing B. distachyon and for
providing wild-type seed (Bd21-3). This study relied upon the
dedicated support of the IBMP gardeners, bioinformatics platform,
and sequencing facility staff. The Blevins Group is supported by the
LabEx consortium ANR-10-LABX-0036_NETRNA (“Investissements d’Avenir”) and by the French Agence Nationale de la
Recherche (ANR) Grant ANR-17-CE20-0004-01.
References
1. Fire A, Xu S, Montgomery MK et al (1998)
Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. https://doi.org/
10.1038/35888
2. Hamilton AJ, Baulcombe DC (1999) A species
of small antisense RNA in posttranscriptional
gene silencing in plants. Science (80-)
286:950–952. https://doi.org/10.1126/sci
ence.286.5441.950
3. Mette MF, Aufsatz W, van der Winden J et al
(2000) Transcriptional silencing and promoter
methylation triggered by double-stranded
RNA. EMBO J 19:5194–5201. https://doi.
org/10.1093/emboj/19.19.5194
4. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J
21:4671–4679.
https://doi.org/10.1093/
emboj/cdf464
5. Xie Z, Johansen LK, Gustafson AM et al
(2004) Genetic and functional diversification
of small RNA pathways in plants. PLoS Biol
2:E104.
https://doi.org/10.1371/journal.
pbio.0020104
6. Onodera Y, Haag JR, Ream T et al (2005)
Plant nuclear RNA polymerase IV mediates
siRNA and DNA methylation-dependent heterochromatin formation. Cell 120:613–622.
https://doi.org/10.1016/j.cell.2005.02.007
7. Vazquez F, Vaucheret H, Rajagopalan R et al
(2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.
Mol Cell 16:69–79. https://doi.org/10.
1016/j.molcel.2004.09.028
8. Zilberman D, Cao X, Johansen LK et al (2004)
Role of Arabidopsis ARGONAUTE4 in
RNA-directed DNA methylation triggered by
inverted repeats. Curr Biol 14:1214–1220.
https://doi.org/10.1016/j.cub.2004.06.055
9. Reinhart BJ, Weinstein EG, Rhoades MW et al
(2002) MicroRNAs in plants. Genes Dev
16:1616–1626.
https://doi.org/10.1101/
gad.1004402
408
Marcel Bo ¨ hrer et al.
when using Klenow probes because of their lower specificity
(Fig. 5). Caution: Ensure that all buffer components are fully
dissolved by preheating the solution to 35
C.
Acknowledgments
M.B., A.G., and D.P. developed the bioinformatics pipeline for TE
annotation, genome browser display, and northern blot probe
design. M.B., B.R., and C.H. performed the molecular genetic
and benchtop experiments. R.S., D. L.-C., A.C., and
J.V. provided B. distachyon mutant germplasm identified using
high-throughput sequencing and bioinformatics; work at the US
DOE Joint Genome Institute is supported by the Office of Science
of the US Department of Energy under Contract no. DE-AC0205CH1123 and by user agreement FP00004794 between JGI and
the USDA Agricultural Research Service. M.B., B.R., and
T.B. assembled the figures and wrote the manuscript. The authors
thank Hugues Renault for advice on culturing B. distachyon and for
providing wild-type seed (Bd21-3). This study relied upon the
dedicated support of the IBMP gardeners, bioinformatics platform,
and sequencing facility staff. The Blevins Group is supported by the
LabEx consortium ANR-10-LABX-0036_NETRNA (“Investissements d’Avenir”) and by the French Agence Nationale de la
Recherche (ANR) Grant ANR-17-CE20-0004-01.
References
1. Fire A, Xu S, Montgomery MK et al (1998)
Potent and specific genetic interference by
double-stranded RNA in Caenorhabditis elegans. Nature 391:806–811. https://doi.org/
10.1038/35888
2. Hamilton AJ, Baulcombe DC (1999) A species
of small antisense RNA in posttranscriptional
gene silencing in plants. Science (80-)
286:950–952. https://doi.org/10.1126/sci
ence.286.5441.950
3. Mette MF, Aufsatz W, van der Winden J et al
(2000) Transcriptional silencing and promoter
methylation triggered by double-stranded
RNA. EMBO J 19:5194–5201. https://doi.
org/10.1093/emboj/19.19.5194
4. Hamilton A, Voinnet O, Chappell L, Baulcombe D (2002) Two classes of short interfering RNA in RNA silencing. EMBO J
21:4671–4679.
https://doi.org/10.1093/
emboj/cdf464
5. Xie Z, Johansen LK, Gustafson AM et al
(2004) Genetic and functional diversification
of small RNA pathways in plants. PLoS Biol
2:E104.
https://doi.org/10.1371/journal.
pbio.0020104
6. Onodera Y, Haag JR, Ream T et al (2005)
Plant nuclear RNA polymerase IV mediates
siRNA and DNA methylation-dependent heterochromatin formation. Cell 120:613–622.
https://doi.org/10.1016/j.cell.2005.02.007
7. Vazquez F, Vaucheret H, Rajagopalan R et al
(2004) Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs.
Mol Cell 16:69–79. https://doi.org/10.
1016/j.molcel.2004.09.028
8. Zilberman D, Cao X, Johansen LK et al (2004)
Role of Arabidopsis ARGONAUTE4 in
RNA-directed DNA methylation triggered by
inverted repeats. Curr Biol 14:1214–1220.
https://doi.org/10.1016/j.cub.2004.06.055
9. Reinhart BJ, Weinstein EG, Rhoades MW et al
(2002) MicroRNAs in plants. Genes Dev
16:1616–1626.
https://doi.org/10.1101/
gad.1004402
408
Marcel Bo ¨ hrer et al.