Pgm t easy vector

We used 5′ and 3′ RACE (rapid amplification of the cDNA ends) to determine the full-length RP11-624L4.1, including the transcriptional initiation and termination sites with a GeneRacer Kit (Invitrogen, USA) according to the manufacturer’s instructions. In brief, total RNA from CNE1 cells was extracted with an RNeasy Mini Kit (QIAGEN), and gDNA was removed by on-column deoxyribonuclease I (ribonuclease [RNase] free; New England Biolabs) digestion. The poly(A) tail detection assay was performed using total RNA from CNE1 cells, which were treated with or without poly(A) polymerase (TAKARA). The relative abundance of TROJAN in poly(A) polymerase-treated or untreated RNA was determined by qPCR. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (containing poly(A)) and U6 (without poly(A)) were used as reference genes. 5′ RACE and 3′ RACE products were amplified with their respective lncRNA-specific primers and cloned into the pGM-T Easy Vector (TIANGEN, P.R. China) for sequencing, and the spliced full-length lncRNAs were obtained using a new pair of primers. The sequences for the gene-specific PCR primers used for 5′ and 3′ RACE analysis are given in the Supplemental Information.

DNA methylation was analyzed using bisulphite sequencing, as previously described [25 (link)]. Approximately 0.5 μg of genomic DNA was treated with sodium bisulfite and subjected to PCR. Subsequently, PCR products were separated on 1% agarose and purified using a TIANgel Midi Purification Kit (Tiangen). Then, the purified PCR products were cloned into a pGMT-Easy vector (Tiangen). For each sample, at least five clones were sequenced for evaluation.