Fig. 3: Identification of the marsupial lncRNA in adult testis and mature sperm.

A Identification of an antisense transcript at the TSS of the tammar MESTA with transcriptome data. Tammar testis transcriptome data was visualised with IGV in a strand specific manner. There was an antisense transcript candidate at the TSS of MESTA in the tammar testis. B Characterisation of the full-length sequence of the antisense lncRNA by RACE reactions. 5′ and 3′ RACE and nested RACE were performed using adult testis with primers for the putative antisense transcript. 5′ RACE generated a single amplicon whereas 3′RACE reactions resulted in three different products. The two 3′RACE products (black asterisks) encoded a non-coding transcript with a polyadenylation signal (red letters) and poly-A tail (green letters). The largest RACE product encoded an isoform of CEP41 with a protein coding sequence (909 bp). C RT-PCR analysis of the marsupial lncRNA in sperm. Sperm samples were collected from three adult males, #1, #2, and #3, and total RNAs were purified from them. cDNAs were synthesised with (RT+) or without reverse transcription (RT−) using the oligo(dT) primer, and PCR was performed with primer pairs for the tammar lncRNA. Presence of the transcript in sperm was confirmed in all biological replicates. M Molecular marker, P Positive control (adult testis), WT Water.