6. If standard Illumina RA3 adapters are detected on the 3
0 -end of
the FASTQ reads, then trim away these adapter sequences
using Cutadapt (options: -a TGGAATTCTCGGG --trim-n -minimum-length 15 --maximum-length 40 -q 30 --discarduntrimmed).
7. Evaluate the trimmed FASTQ reads by re-running FastQC on
the Cutadapt output files.
8. Map the trimmed small RNA-seq reads to the reference
genome (here: B. distachyon Bd21 v3.0) and tally small RNAs
within annotated TEs using ShortStack (options: --nohp -dicermin 20 --dicermax 25). ShortStack will invoke the Bowtie
read aligner and call SAMtools (see Note 4).
9. Classify the annotated LTR TEs in a tab-delimited file, sorted
with TEs showing the highest levels of 24 nt siRNAs first,
allowing the identification of putative Pol IV targets within
the B. distachyon genome (TE-siRNA clusters).
10. Verify selected TE-siRNA clusters by loading sorted and
indexed BAM files from step 8 into either JBrowse [57] or
Integrative Genomics Viewer [58] and viewing siRNA clusters
along the B. distachyon chromosomes, focusing on LTR TE
loci with the most abundant 24 nt siRNAs. Both de novo TE
(step 2) and community TE/gene annotations can be
integrated to support this analysis.
11. Curate promising candidates by hand, using Geneious Prime
to annotate the LTRs, target-site duplications, tRNA primerbinding sites, polypurine tracks, and ORFs that could indicate
whether individual LTR TEs are potentially functional. Functional TEs typically have intact ORFs and perfectly identical or
nearly identical LTRs.
12. Finally, select LTR TEs with abundantly mapped 24 nt siRNAs
for validation by small RNA northern blot. Very copious individual siRNA sequences can be used to design complementary
DNA oligo probes for radioactive 5
0 -end-labeling. Otherwise,
TE subfeatures displaying many distinct siRNA mappings, such
as ~600 bp spanning an LTR, can be used to design PCR
amplicons for Klenow probe labeling (Fig. 3).
3.2 Extraction of
Total RNA from Plant
Tissue
Wear a lab coat and gloves for all the following procedures. The
pipettes, tips, and tubes should all be kept isolated and clean before
RNA extraction. In addition, filter tips are recommended to avoid
RNase contamination and degradation of the final RNA samples.
1. Grind plant tissue samples to a fine powder in liquid nitrogen
using a pre-cooled mortar and pestle (see Note 5).
2. Transfer about 400 μL of this powder from each sample to a
13 mL round-bottomed tube (kept in liquid nitrogen) (see
Note 6).
Genome-Scale and Northern Blot Analyses of siRNAs
397
0 -end of
the FASTQ reads, then trim away these adapter sequences
using Cutadapt (options: -a TGGAATTCTCGGG --trim-n -minimum-length 15 --maximum-length 40 -q 30 --discarduntrimmed).
7. Evaluate the trimmed FASTQ reads by re-running FastQC on
the Cutadapt output files.
8. Map the trimmed small RNA-seq reads to the reference
genome (here: B. distachyon Bd21 v3.0) and tally small RNAs
within annotated TEs using ShortStack (options: --nohp -dicermin 20 --dicermax 25). ShortStack will invoke the Bowtie
read aligner and call SAMtools (see Note 4).
9. Classify the annotated LTR TEs in a tab-delimited file, sorted
with TEs showing the highest levels of 24 nt siRNAs first,
allowing the identification of putative Pol IV targets within
the B. distachyon genome (TE-siRNA clusters).
10. Verify selected TE-siRNA clusters by loading sorted and
indexed BAM files from step 8 into either JBrowse [57] or
Integrative Genomics Viewer [58] and viewing siRNA clusters
along the B. distachyon chromosomes, focusing on LTR TE
loci with the most abundant 24 nt siRNAs. Both de novo TE
(step 2) and community TE/gene annotations can be
integrated to support this analysis.
11. Curate promising candidates by hand, using Geneious Prime
to annotate the LTRs, target-site duplications, tRNA primerbinding sites, polypurine tracks, and ORFs that could indicate
whether individual LTR TEs are potentially functional. Functional TEs typically have intact ORFs and perfectly identical or
nearly identical LTRs.
12. Finally, select LTR TEs with abundantly mapped 24 nt siRNAs
for validation by small RNA northern blot. Very copious individual siRNA sequences can be used to design complementary
DNA oligo probes for radioactive 5
0 -end-labeling. Otherwise,
TE subfeatures displaying many distinct siRNA mappings, such
as ~600 bp spanning an LTR, can be used to design PCR
amplicons for Klenow probe labeling (Fig. 3).
3.2 Extraction of
Total RNA from Plant
Tissue
Wear a lab coat and gloves for all the following procedures. The
pipettes, tips, and tubes should all be kept isolated and clean before
RNA extraction. In addition, filter tips are recommended to avoid
RNase contamination and degradation of the final RNA samples.
1. Grind plant tissue samples to a fine powder in liquid nitrogen
using a pre-cooled mortar and pestle (see Note 5).
2. Transfer about 400 μL of this powder from each sample to a
13 mL round-bottomed tube (kept in liquid nitrogen) (see
Note 6).
Genome-Scale and Northern Blot Analyses of siRNAs
397