Figure 1. Identification and validation of lncRNAs in D. melanogaster.

(a) Analytical diagram for Drosophila lncRNA filtering and prediction. The transcriptome database of D. melanogaster contains an annotation of 43,967 transcripts. All transcripts were subject to three-step filterings and a computational prediction of ncRNAs using the program CPAT. False positive rate of lncRNA prediction was evaluated based on protein-coding gene annotations in Flybase. (b) Length distribution of the 976 lncRNAs identified in D. melanogaster. (c) Validation of 20 predicted lncRNAs using RT-PCR. The cDNA transcripts were transcribed from RNA with reverse transcriptase, and then used as PCR templates. PCR templates were genomic DNA in the positive control. In the negative control, RNA samples were subject to the same reverse transcription process with omission of the reverse transcriptase. Lanes on the two sides of gel are 100 DNA ladder. See details about the 20 lncRNAs in Supplementary Table S1 and Supplementary data 1. (d) Validation of 12 predicted intergenic lncRNAs by RNA dot blot. The 12 lncRNAs are among the 20 lncRNAs and had been validated by RT-PCR. Total 10 μg RNA were hybridized with Biotin labelled probes on nylon membrane. The 12 lncRNAs are as follows: chr3L_18316644_18317098, chr2L_18795816_18796184, chr3LHet_2236180_2236548, chr2L_21798044_21799854, chr3RHet_1825840_1826355, MIP19078, chrX_3968951_3969506, chr3L_18686588_18686941, chr2L_3268204_3268702, chr2L_8887436_8888049, hr2L_22705365_22706170, chr2R_135227_135517 (also see Supplementary Table S1). (e) Example of a validated novel lncRNA candidate, i.e., chr2R_135227_135517. The chromosome coordinate based on Flybase version FB2014_06 is shown within a range of approximately 30 kilo-bp. The lncRNA indicated by red arrow neighbours coding gene IntS3. Bottom colourful tracks show the expression of transcripts from RNA-seq reads in 27 developmental stage, from 0–2-h embryos to 30-d adults.