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. 2014 Jul 23;43(1):e1. doi: 10.1093/nar/gku637

Figure 2.

Figure 2.

Detailed LQ cloning method. (A) A pre-adenylated (rApp) 3′-terminal dideoxy-C (ddC) blocked adapter (gray) is annealed to a ssDNA reverse transcription (RT) oligo (black) in a 1:1 molar ratio. The annealed adapter is ligated to 3′-hydroxyl-containing RNA (orange) using T4 RNA Ligase 2 (truncated K227Q) without ATP. Each RT oligo contains a 5′ Guanine (G) followed by a 4 or 6 nucleotide randomer (NX), a 3–6 nucleotide barcode (BAR) and 3 internal deoxyUridine (dU) nucleotides. The adapter::RT oligo hybrid is in excess over RNA, resulting in free adapter::primer material present in the completed reaction. (B) Reverse transcription of ligated RNA is carried out in the same tube as the ligation reaction generating ‘+ insert’ and ‘no insert’ cDNA products (red and black line) using dGTP, dTTP, dATP, dCTP as well as biotinylated dATP and dCTP (yellow ‘B’-containing circles). The RNA template is degraded (dashed orange line) by base hydrolysis and cDNA is ethanol precipitated with ammonium acetate to facilitate maximum removal of free adapter and unincorporated nucleotides (C). Ethanol precipitated cDNAs are circularized (D) and resolved on a 10% denaturing polyacrylamide gel. ‘+ insert’ circularized cDNAs are isolated by excising and eluting them from the gel overnight in the presence of magnetic streptavidin beads (E). Bead-bound ‘+ insert’ cDNAs serve as templates in the first round of PCR. Amplification is done using a mix containing uracil-N-deglycosylase (UNG) to remove dU nucleotides, thereby generating a linear template through strand scission, and with primers complimentary to the 3′ adapter (blue) and 5′ end of the RT oligo (tan) (F). First round PCR products are resolved on an 8% native polyacrylamide gel, the 60–70 nucleotide products are excised and a portion is used as the template for second round PCR. Second round PCR products are generated using primers complimentary to the 3′ adapter (dark blue) and 5′ end of the RT oligo (brown) that contain the full Illumina or Ion Torrent adapter sequences (dark blue and brown) (G).