Targeted Sequencing of TIN2 transcripts

TIN2 is an important regulator of telomere length, and mutations in TINF2, the gene encoding TIN2, cause short telomere syndromes. To identify full-length transcripts from the TINF2 gene in human and mouse cells, we used a modified 3’ RACE paired with PacBio sequencing. Whole cell RNA was reverse transcribed using oligo(dT20) with an adapter sequence, then TIN2 mRNA was specifically amplified using primers to the adapter sequence and to a unique sequence in exon 1. These amplicons were sequenced with PacBio sequencing to determine the splicing variants, and we were able to identify a novel isoform expressed in human but not mouse cells. This isoform may have been missed by previous sequencing approaches because it does not contain a novel splice junction but rather a unique pattern of spliced and retained introns.

TIN2是端粒长度的重要调控因子，编码TIN2的基因TINF2发生突变可引发短端粒综合征。为鉴定人类与小鼠细胞中TINF2基因的全长转录本，我们采用改良的3'末端快速扩增技术（3’ RACE）结合PacBio测序的策略。我们使用带有接头序列的oligo(dT)20引物对全细胞RNA进行反转录，随后利用针对接头序列与外显子1中独特序列的引物，特异性扩增TIN2 mRNA。将上述扩增产物通过PacBio测序进行测序以确定其剪接变体，最终我们在人类细胞中鉴定到一种新型异构体，而小鼠细胞中未检测到该异构体。该异构体之所以可能被此前的测序方法遗漏，是因为它并不含有新型剪接接头，而是呈现出独特的剪接与内含子保留模式。