4 protocols
nucleic acid extraction protocol
Human polyA+ RNAs were purchased from Clontech. The RNAs were isolated from tissue samples using a modified guanidium thiocynate extraction method followed by polyA+ RNA selection with two rounds of oligo(dT)-cellulose columns.
nucleic acid sequencing protocol
The emulsion PCRs (emPCRs) for the library titrations and the sequencing reactions were prepared according to the emPCR Method Manual - Lib-L LV for GS FLX+ Series - XL+. Sequencing was performed on a GS FLX+ Instrument following the Sequencing Method Manual for GS FLX+ Series / XL+ Kit. Sequencing images were analyzed with the Roche 454 Shotgun-pipeline program and then a FastQ file was generated.
nucleic acid library construction protocol
Libraries were prepared according to the Rapid Library Preparation Method Manual for GS FLX+ Series / XL+ Kit.
Rapid Amplification of cDNA Ends
5' and 3' RACE reactions were performed in 12.5 ul volume independently with each polyA+ RNA sample using the SMART RACE amplification kit (Clontech) according to the manufacturer’s instructions. 7.5ul of each of the 400 lncRNA targets were pooled respecting tissues and directionality of the RACE reaction (5' and 3' RACE) and purified with the QIAquick kit (Quiagene) as described for RT-PCR-seq experiments [2]. A set of 25 control reactions targeting full-length annotated transcripts were RACEd in parallel as positive controls. 1.5ul of these controls were pooled with the other reactions prior to library preparation.