regression model based on four sequence-based features:
ORF size, ORF coverage, Fickett Score, and Hexamer
usage bias. A cutoff of coding potential (CP) score < À0.5
can be used to distinguish coding RNAs from
noncoding RNAs.
13. Putative lncRNAs that passed the filters on steps 11 and 12 can
be annotated to the reference genome using PAVIS webtools
in the website https://manticore.niehs.nih.gov/pavis2/anno
tate. Species of interest and length of upstream and downstream regions should be selected. A strand-specific BED file
containing chromosomal locations for the lncRNAs is the
input required for the analysis. The output classifies each
lncRNA region according to its genomic context in upstream,
5
0 UTR, exon, intron, 3
0 UTR, downstream, and intergenic,
similar to the classifications shown in Fig. 1. Alternatively, a
pipeline known as PLAR (Pipeline for lncRNA annotation
from RNA-seq data; http://www.weizmann.ac.il/Biological_
Regulation/IgorUlitsky/PLAR) can be used to annotate
lncRNAs [56]. Comparison to known databases of lncRNAs
and a final manual inspection in a genome browser (i.e., IGV) is
recommended to check location and strand specificity for new
lncRNAs.
4 Notes
1. Good results have been obtained using the monoclonal ANTIFLAG® M2 antibody produced in mouse (Sigma-Aldrich,
www.sigmaaldrich.com, cat. # F1804).
2. Use a mask for lung protection while weighing the following
reagents: Tris base, EGTA, Brij-35, DTT, PMSF, cycloheximide, and chloramphenicol.
3. Take EGTA to the desired pH with NaOH pellets instead of
solutions or highly concentrated solutions. Diluted NaOH
solutions can absorb carbon dioxide from the air over time,
which lowers the pH, producing the precipitation of EGTA.
4. Pipette detergents with a 1000 μL tip enlarged by cutting
0.5 cm from the end. It takes time for the detergents to go
into solution. Add heparin and RNase inhibitor to PEB if
working with tissue with high RNase content such as mature
maize leaves. When working with seeds or mature leaves
improve the disruption of ribosome-cytoskeleton association
by including 1% (w/v) sodium deoxycholate DOC, prepared
from a 20% (w/v) stock solution, in the PEB. Use freshly
prepared solutions including cycloheximide and DTT.
TRAP-SEQ Analysis of Long Noncoding RNAs
467
ORF size, ORF coverage, Fickett Score, and Hexamer
usage bias. A cutoff of coding potential (CP) score < À0.5
can be used to distinguish coding RNAs from
noncoding RNAs.
13. Putative lncRNAs that passed the filters on steps 11 and 12 can
be annotated to the reference genome using PAVIS webtools
in the website https://manticore.niehs.nih.gov/pavis2/anno
tate. Species of interest and length of upstream and downstream regions should be selected. A strand-specific BED file
containing chromosomal locations for the lncRNAs is the
input required for the analysis. The output classifies each
lncRNA region according to its genomic context in upstream,
5
0 UTR, exon, intron, 3
0 UTR, downstream, and intergenic,
similar to the classifications shown in Fig. 1. Alternatively, a
pipeline known as PLAR (Pipeline for lncRNA annotation
from RNA-seq data; http://www.weizmann.ac.il/Biological_
Regulation/IgorUlitsky/PLAR) can be used to annotate
lncRNAs [56]. Comparison to known databases of lncRNAs
and a final manual inspection in a genome browser (i.e., IGV) is
recommended to check location and strand specificity for new
lncRNAs.
4 Notes
1. Good results have been obtained using the monoclonal ANTIFLAG® M2 antibody produced in mouse (Sigma-Aldrich,
www.sigmaaldrich.com, cat. # F1804).
2. Use a mask for lung protection while weighing the following
reagents: Tris base, EGTA, Brij-35, DTT, PMSF, cycloheximide, and chloramphenicol.
3. Take EGTA to the desired pH with NaOH pellets instead of
solutions or highly concentrated solutions. Diluted NaOH
solutions can absorb carbon dioxide from the air over time,
which lowers the pH, producing the precipitation of EGTA.
4. Pipette detergents with a 1000 μL tip enlarged by cutting
0.5 cm from the end. It takes time for the detergents to go
into solution. Add heparin and RNase inhibitor to PEB if
working with tissue with high RNase content such as mature
maize leaves. When working with seeds or mature leaves
improve the disruption of ribosome-cytoskeleton association
by including 1% (w/v) sodium deoxycholate DOC, prepared
from a 20% (w/v) stock solution, in the PEB. Use freshly
prepared solutions including cycloheximide and DTT.
TRAP-SEQ Analysis of Long Noncoding RNAs
467