Poly(A)+, poly(A)- and bimorphic transcripts revealed by deep sequencing. (a) A diagram of the experimental approach. Total RNAs were extracted from H9 cells or HeLa cells and treated with DNaseI before being subjected to poly(A)+ and poly(A)- transcript enrichment. See text for details. The enriched poly(A)- and poly(A)+ RNAs were used to prepare single-end RNA-Seq libraries. The size-selected single-end libraries were sequenced using 76 cycles. The single-end reads were trimmed from the 3' end to a total length of 75 nucleotides prior to alignment. (b) Agarose gel electrophoresis to confirm the poly(A)- RNA purification. The gel on the left shows that the poly(A)+ RNA fraction from HeLa cells contains no detectable rRNA but that the poly(A)- material not bound to oligo(dT) beads contained most of the rRNA. The gel on the right shows that subsequent rRNA depletion removes the great majority of rRNA from the poly(A)- sample. M, the molecular weight marker. (c) A diagram of the analytical approach. Sequence analysis involved aligning all reads to a combined database of the genome and splice junctions using Bowtie [15,19]. The read counts were then further analyzed using the normalized value BPKM (bases per kilobase of gene model per million mapped bases) to identify poly(A)- and bimorphic transcripts that were significantly different between the poly(A)+ and poly(A)- samples. (d) Classification of poly(A)+, poly(A)- and bimorphic predominant transcripts. Poly(A)+, poly(A)- and bimorphic predominant transcripts were classified according to their relative abundance between the poly(A)+ and poly(A)- samples in individual cell lines. See text and Materials and methods for details.
Yang et al. Genome Biology 2011 12:R16 doi:10.1186/gb-2011-12-2-r16