The linkered cDNA pool from each individual sample was amplified for 20 cycles [as described in (12 (link))] with an unique pair of barcoded primers. A second round of amplification with temperatures and cycle lengths identical to the initial round, but with variable number of total cycles (six, eight and ten), was performed using 1/25th of the first PCR product, dNTPs, barcoded primers, buffer and Taq DNA polymerase in a 40 μl PCR mix. An analytical, non-denaturing 4% Nusieve gel was used to visually determine the fewest number of cycles that yielded a visible signal for PCR products containing inserts corresponding to small RNAs. DNA was extracted from the gel using the Qiaquick gel extraction kit (Qiagen). Although the instructions for gel extraction called for dissolution of the gel slice in Buffer QG at 50°C, we performed all manipulations of the DNA-containing buffers at room temperature, to ensure that the complex PCR mixtures were not denatured.
To obtain a rough quantitation of individual libraries, 10% of each recovered PCR product was separated on an analytical 2% agarose gel. Based on the intensities of bands on this gel, PCR products from 25 samples were pooled together in roughly equimolar ratios. Quality of the mixture was tested by conventional Sanger sequencing of 46 PCR products that had been cloned into TOPO-TA vectors (Invitrogen). If the sequence quality was satisfactory, 300 ng of the mixture was run on a non-denaturing 2% low melting point (LMP)-agarose gel (run I) or on a denaturing 6% PAGE–urea gel (run II; recommended) for quantification, and for further size-restricted purification of barcoded species that contain small RNA inserts. DNA was eluted from the PAGE–urea gel overnight at 4°C in 0.3 M NaCl.
To obtain a rough quantitation of individual libraries, 10% of each recovered PCR product was separated on an analytical 2% agarose gel. Based on the intensities of bands on this gel, PCR products from 25 samples were pooled together in roughly equimolar ratios. Quality of the mixture was tested by conventional Sanger sequencing of 46 PCR products that had been cloned into TOPO-TA vectors (Invitrogen). If the sequence quality was satisfactory, 300 ng of the mixture was run on a non-denaturing 2% low melting point (LMP)-agarose gel (run I) or on a denaturing 6% PAGE–urea gel (run II; recommended) for quantification, and for further size-restricted purification of barcoded species that contain small RNA inserts. DNA was eluted from the PAGE–urea gel overnight at 4°C in 0.3 M NaCl.
