(c) Experimental design (including samples, replicates and
different treatments, etc.)
(d) Normalization and statistical test methods (a sizeFactor,
i.e., a scaling coefficient that applies to raw counts, and a
normalization factor, i.e., a scaling coefficient that applies
to library sizes.)
(e) Filtering process (setting p value and lowest count reads)
For detailed usage of SARTools, the user manual can be
found online (Table 3). Based on the tables, the expression of
specific sRNAs of interest can be further analyzed. Clustering
analysis such as heatmap (http://biit.cs.ut.ee/clustvis/), Gene
Ontology (GO) enrichment analysis (http://geneontology.
org/docs/go-enrichment-analysis/), and others can be used
according to different experimental purposes.
4 Notes
1. This protocol is designed for 50 nM adapter oligos.
Corresponding recalculation is needed for different amount
of adapter oligos.
2. The 3
0 end modification of the 3
0 adapter used here is a NH 2 . A
dideoxy nucleotide (ddC) is also a good alternative and available in numerous vendors.
3. For RNA markers and adapters, and primers that are directly
added to RNA, use RNase-free water to dissolve them, and the
following solution (e.g., 0.4 M NaCl) should also be RNasefree. Other index primers or SR primer can be dissolved by
autoclaved double-distilled water.
4. Dicing gel into smaller pieces, the higher recycle efficiency will
be obtained. But if the pieces are too small, it might be difficult
to pick up. Researchers need to make a balance here.
5. For the laboratories which do not have permission for radioisotope usage, initial separation of sRNAs could be achieved
through resolving total RNA samples align with 19- and 24-nt
oligo markers that are prepared in the same volume and buffer
compositions so that sRNAs and markers could migrate at the
identical speed in a gel. In this scenario, one could just conduct
separation of sRNA cDNAs at the final step as instructed by
most of commercial kits, and does not need to monitor the 3
0
and 5
0 ligation efficiency step by step.
6. If oligo markers are synthesized by companies, the default
synthesis is that the 5
0 end contains a hydroxyl group. Under
this substance, no CIP treatment is needed. If the markers
Identification and Quantification of sRNAs
251
different treatments, etc.)
(d) Normalization and statistical test methods (a sizeFactor,
i.e., a scaling coefficient that applies to raw counts, and a
normalization factor, i.e., a scaling coefficient that applies
to library sizes.)
(e) Filtering process (setting p value and lowest count reads)
For detailed usage of SARTools, the user manual can be
found online (Table 3). Based on the tables, the expression of
specific sRNAs of interest can be further analyzed. Clustering
analysis such as heatmap (http://biit.cs.ut.ee/clustvis/), Gene
Ontology (GO) enrichment analysis (http://geneontology.
org/docs/go-enrichment-analysis/), and others can be used
according to different experimental purposes.
4 Notes
1. This protocol is designed for 50 nM adapter oligos.
Corresponding recalculation is needed for different amount
of adapter oligos.
2. The 3
0 end modification of the 3
0 adapter used here is a NH 2 . A
dideoxy nucleotide (ddC) is also a good alternative and available in numerous vendors.
3. For RNA markers and adapters, and primers that are directly
added to RNA, use RNase-free water to dissolve them, and the
following solution (e.g., 0.4 M NaCl) should also be RNasefree. Other index primers or SR primer can be dissolved by
autoclaved double-distilled water.
4. Dicing gel into smaller pieces, the higher recycle efficiency will
be obtained. But if the pieces are too small, it might be difficult
to pick up. Researchers need to make a balance here.
5. For the laboratories which do not have permission for radioisotope usage, initial separation of sRNAs could be achieved
through resolving total RNA samples align with 19- and 24-nt
oligo markers that are prepared in the same volume and buffer
compositions so that sRNAs and markers could migrate at the
identical speed in a gel. In this scenario, one could just conduct
separation of sRNA cDNAs at the final step as instructed by
most of commercial kits, and does not need to monitor the 3
0
and 5
0 ligation efficiency step by step.
6. If oligo markers are synthesized by companies, the default
synthesis is that the 5
0 end contains a hydroxyl group. Under
this substance, no CIP treatment is needed. If the markers
Identification and Quantification of sRNAs
251