Journal: Nucleic Acids Research
Article Title: Poly(A)-ClickSeq: click-chemistry for next-generation 3΄-end sequencing without RNA enrichment or fragmentation
Figure Lengend Snippet: Schematic overview of Poly(A)ClickSeq (PAC-seq). ( A ) RT-PCR is launched from a non-anchored Poly(T) primer containing a portion of the Illumina p7 adaptor. RT-PCR is performed in the presence of AzATP, AzGTP and AzCTP, but not AzTTP, thus only allowing chain termination to occur upstream of the poly(A) tail in the 3΄UTR. ( B ) 3΄-Azido-blocked cDNA fragments are ‘click-ligated’ to 5΄-hexynyl–functionalised DNA oligos containing the p5 Illumina adaptor. This yields triazole-linked ssDNA which can be PCR-amplified using primers to the p5 and p7 Illumina adaptors. ( C ) The cDNA library is analysed by gel electrophoresis and should consist of a smear of DNA products centered ∼200–300 bp. Appropriate cDNA fragment sizes are cut out of the gel and purified to yield a final library. ( D ) The final library consists of DNA fragments containing the Illumina p5 adaptor, a portion of the 3΄UTR, a stretch of As derived from both the RNA template and the poly(T) primer, and finally the p7 Illumina Indexing primer.
Article Snippet: Optimized thermocycler conditions are as follows: 94° 4 min; 53° 30 s; 68° 10 min; [94° 30 s, 53° 30 s, 68° 2 min] × 20–22; 68° 5 min. Amplified PCR product was then run on a 2% precast agarose e-gel (Invitrogen, E-Gel Electrophoresis System) for 10 min and ∼200–300 bp fragments (for 1 × 150 SE Illumina) or ∼200–400 bp fragments (for 1 × 250 SE Illumina) were excised and cleaned using the Zymo Research Gel DNA Recovery Kit.
Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, cDNA Library Assay, Nucleic Acid Electrophoresis, Purification, Derivative Assay