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An RNA-Seq Strategy to Detect the Complete Coding and Non-Coding Transcriptome Including Full-Length Imprinted Macro ncRNAs

Figure 1

Optimisation and reproducibility of ribo-depleted RNA-Seq.

(A) Distribution of different sequence tag types from RNA prepared from CCE differentiated ES cells subject to ribosomal RNA depletion using either the RiboMinus or the Ribo-Zero Kit and fragmented either by RNA-hydrolysis or by cDNA-shearing. Sequencing was performed in two different sequencing locations (Vienna-IMP, Nijmegen, RiboMinus) or in one sequencing location (Vienna-CeMM, Ribo-Zero). The percentage of tags in each category is shown for two technical sequencing replicates (CCE1, CCE2) of material prepared by RiboMinus and cDNA-shearing (sheared, lanes nr. 1,2,5,6) or RiboMinus and RNA-hydrolysis (hydrolysed, lanes nr. 3,4,7,8), for the combination of three technical sequencing replicates of RiboMinus and RNA-hydrolysis (lane nr. 9) and for one sequencing of Ribo-Zero and RNA-hydrolysis (lane nr. 10). green: unique tags matching only once in the genome; blue: rRNA+mitoRNA tags matching to ribosomal (RiboMinus and Ribo-Zero) or mitochondrial (RiboMinus) RNAs; red: repeat tags matching more than once in the genome; purple: nomatch tags do not match to the genome. (B) Scatter plots comparing the RPKM (Reads Per Kilobase of exon model per Million of reads) transcription levels of RefSeq protein-coding genes between combined tags from RiboMinus and RNA-hydrolysis (H) and RiboMinus and cDNA-shearing (S) from CCE within the same location: Vienna-IMP (left) and Nijmegen (right). (C) Scatter plots as in B comparing RPKM transcript levels of all combined tags from the two sequencing locations (Vienna-IMP and Nijmegen, left) or between the combined RiboMinus data and the Ribo-Zero data (right). R: Pearson's correlation, note that a perfect correlation is R = 1.

Figure 1