a polyacrylamide gel. These size-separated small RNAs are then
transferred to a nylon membrane. Cross-linking is performed to
fix the RNA to the membrane, allowing successive hybridizations
and detections using radiolabeled probes. One difficulty with this
technique is its relatively low sensitivity compared to small
RNA-seq. Even using sensitive phosphor-imaging systems, hybridization probes must be carefully designed and the hybridization
procedure optimized to detect plant siRNAs, because these molecules can accumulate at orders of magnitude lower levels than
miRNAs [47].
Here, we present an integrated protocol to study small RNA
pathways and TE silencing in organisms that lack comprehensive,
consolidated TE annotations, such as the model grass Brachypodium distachyon. We combined publicly available small RNA-seq
data and standard bioinformatics tools to de novo annotate the
B. distachyon genome (with both TEs and siRNA clusters), and
then performed northern blot validation of siRNA source loci
overlapping TEs. To this end, we developed a software pipeline
that identifies long terminal repeats (LTRs), combines these
regions with published TE annotations, and cross-compares all
annotations to the mapped positions of siRNAs (Fig. 3). Validation
was performed using an optimized northern blot hybridization
procedure, with the goal of detecting Pol IV-dependent 24 nt
siRNAs in both B. distachyon (monocot) and A. thaliana (eudicot)
plant species.
2 Materials
2.1 Plant Materials
1. Arabidopsis thaliana wild-type (WT, ecotype Col-0) and pol IV
null mutant (At nrpd1-3) [6] plants grown with a day/night
cycle of 16 h/8 h at 21
C/18
C.
2. Brachypodium distachyon WT (inbred line Bd21-3) and a pol IV
point mutant (Bd nrpd1-1) in the Bd21-3 background grown
with a day/night cycle of 20 h/4 h at 25
C.
3. RNA extractions from inflorescences of A. thaliana or from
young leaves of B. distachyon. Plant materials harvested, flash
frozen in liquid nitrogen, and stored at À80
C.
2.2 Bioinformatics
Analyses
1. Brachypodium distachyon Bd21v3.0 reference genome
assembly (FASTA format) obtained from Phytozome
(GCF_000005505.3_Brachypodium_distachyon_v3.0)
[48, 49].
2. Publicly available small RNA-seq data from B. distachyon used
for the analyses (GEO: GSM1266842, BDI09 sample from
leaves) [50].
Genome-Scale and Northern Blot Analyses of siRNAs
391
transferred to a nylon membrane. Cross-linking is performed to
fix the RNA to the membrane, allowing successive hybridizations
and detections using radiolabeled probes. One difficulty with this
technique is its relatively low sensitivity compared to small
RNA-seq. Even using sensitive phosphor-imaging systems, hybridization probes must be carefully designed and the hybridization
procedure optimized to detect plant siRNAs, because these molecules can accumulate at orders of magnitude lower levels than
miRNAs [47].
Here, we present an integrated protocol to study small RNA
pathways and TE silencing in organisms that lack comprehensive,
consolidated TE annotations, such as the model grass Brachypodium distachyon. We combined publicly available small RNA-seq
data and standard bioinformatics tools to de novo annotate the
B. distachyon genome (with both TEs and siRNA clusters), and
then performed northern blot validation of siRNA source loci
overlapping TEs. To this end, we developed a software pipeline
that identifies long terminal repeats (LTRs), combines these
regions with published TE annotations, and cross-compares all
annotations to the mapped positions of siRNAs (Fig. 3). Validation
was performed using an optimized northern blot hybridization
procedure, with the goal of detecting Pol IV-dependent 24 nt
siRNAs in both B. distachyon (monocot) and A. thaliana (eudicot)
plant species.
2 Materials
2.1 Plant Materials
1. Arabidopsis thaliana wild-type (WT, ecotype Col-0) and pol IV
null mutant (At nrpd1-3) [6] plants grown with a day/night
cycle of 16 h/8 h at 21
C/18
C.
2. Brachypodium distachyon WT (inbred line Bd21-3) and a pol IV
point mutant (Bd nrpd1-1) in the Bd21-3 background grown
with a day/night cycle of 20 h/4 h at 25
C.
3. RNA extractions from inflorescences of A. thaliana or from
young leaves of B. distachyon. Plant materials harvested, flash
frozen in liquid nitrogen, and stored at À80
C.
2.2 Bioinformatics
Analyses
1. Brachypodium distachyon Bd21v3.0 reference genome
assembly (FASTA format) obtained from Phytozome
(GCF_000005505.3_Brachypodium_distachyon_v3.0)
[48, 49].
2. Publicly available small RNA-seq data from B. distachyon used
for the analyses (GEO: GSM1266842, BDI09 sample from
leaves) [50].
Genome-Scale and Northern Blot Analyses of siRNAs
391