GENCODE Batch VIII a: determining lncRNA transcript 5' and 3'-ends by RACE-PCR / 454 sequencing

As part of the ENCODE consortium the GENCODE project is producing a reference gene set through manual and automated gene prediction. In the current phase of ENCODE we have found strong evidence that many lncRNAs transcript termini are still unknown. This experiment aims to set up an experimental validation strategy to accurately determine the 5' and 3' ends of transcripts, which is based on semi-nested RACE extensions of annotated 5' and 3' ends followed by high throughput sequencing. A total of 400 highly expressed lncRNA transcript models from Gencode 7 which did not have any CAGE/PET support were selected as the test set whereas 25 transcripts with transcript start site (TSS) supported by CAGE tags and transcript termination site (TTS) supported by PET ditags formed the positive control set. Transcript ends were amplified by RACE-PCR from brain and testis RNA samples and sequenced using the Roche 454 platform. The sequencing was performed at the Andalusian Human Genome Sequencing Centre (CASEGH), Seville, Spain.