Supplementary Figure 4: Strategy for designing and applying multiple probes in a single capture reaction. | Nature Protocols

Supplementary Figure 4: Strategy for designing and applying multiple probes in a single capture reaction.

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

Supplementary Figure 4

(a) An example of designing tiling probes to be pooled together into one capture tube to detect multiple RNA products from a 1.2 Kb region. At the start, 36 tiling probes were selected and scored by applying the OligoWiz1 probe design software. Each probe was scored based on potential cross-hybridization (self-dimer2), melting temperature, potential folding (stem-loop), relative position to the target, and sequence complexity. Top probes selected by OligoWiz were filtered by AutoDimer to remove probes with high potential for cross-reactivity. The final set of probes can be pooled together to capture multiple RNA targets. b) Results for capturing of probes described in (a). Both blank regions (i.e. with probes but no RNA captured) and ultra-deep (over 2.2 million mapped reads) region were detected, suggesting that real transcription of novel RNA can be reliably detected by the TEsR approach. c) Capture of GAPDH from the total RNA sample of the human T19 cell line. The RNA was prepared by the mirVana microRNA Isolation Kit (Ambion) according to the manufacturer’s instructions. Before TEsR capture, 2.5 μg purified total RNA sample was treated to remove ribosomal RNAs by using Ribo-Zero Gold rRNA removal method (Epicentre). Both probes shown in panel 5 were applied in one capturing reaction. Probe 1 was designed to span an exon-exon junction. All of the capture mapped reads shown in panel 4 lie within the exons of the sense GAPDH and the antisense lncRNA. The numbers of reads mapped to the sense (green) and the antisense (purple) RNAs are shown separately in panel 6, with 48512 and 7943 tags per million respectively. Panel 1 shows chromosome 12 ideogram with the GAPDH locus marked by a red bar. Panel 2 and 3 show respectively reference genes and transcripts from Entrez gene, hg19 annotation and Gencode v19 annotation, with antisense long noncoding RNA (lncRNA NST00000602946.1) in purple, and sense messenger RNA for GAPDH in green.

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