Supplementary Figure 5: Protocol steps: bait, adenylated 3′ adapter and Bioanalyzer profiles. | Nature Protocols

Supplementary Figure 5: Protocol steps: bait, adenylated 3′ adapter and Bioanalyzer profiles.

From: Target-enrichment sequencing for detailed characterization of small RNAs

Supplementary Figure 5

(a) Validation of RNA products post reverse transcription. Baits (Bait) were run in a 1% Agarose gel. RNase-treated baits (PCR temp) were used as a positive control, proving that the signal obtained was mainly coming from RNA molecules. (b) Testing the production of adenylated 3’ adapter. ‘Linker’ lane is the control with template only, without adenylation; and the ‘App Linker’ indicates the adenylated adapter. The quality of adenylated adapter product can be judged based on the percent of adenylated vs not adenylated. (c) The top and low panels show the bionanalyzer profiles of precaptured libraries before and after the PCR step respectively. Green boxes indicate the lower Bioanalyser marker (1) and other oligos (2). In particular, (2) -in the upper panel- shows the oligos used for ligation to RNA as well as those used for reverse transcription, however the contamination of undesired oligos (like adapter dimers) after PCR amplification of cDNA is markedly reduced as seen in (2) in the lower panel. Blue boxes show library size distributions and Purple boxes show the upper Bioanalyser marker. The lower bioanalyser profile represents a typical profile described at Step 76. The peak around 100bp corresponds to the precaptured pool of RNAs of microRNA-like length (consider that both 3’ and 5’ linkers together account for approximately 70bp). The other (lower abundant) peak at around 150 bp corresponds to other sRNAs present in the starting RNA samples.

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