To generate double stranded DNA, with the appropriate adaptors for Illumina sequencing, as well as addition of a four nucleotide EMOTE barcode which serves to identify the RNA sample and pool, a PCR reaction was prepared for each RT-reaction: 10 μl PCR-purified RT-reaction, 27 μl water, 10 μl Q5 Polymerase buffer, 1.5 μl dNTP (2.5 mM each), 1.5 μl 100nM of one of primers D6A, D6B, D6C, etc. (each with a unique EMOTE barcode, Table 2 ), 1.5 μl 10 uM Primer B-PE-PCR20, and 0.5 μl Q5® Hot Start High-Fidelity DNA Polymerase (NEB).
These 50 μl PCR reactions with low D6x primer concentration was run for the 5 first cycles of the following program: 2 min @98 °C, (10s @98 °C, 20s @50 °C, 2 min @ 72 °C) 31 cycles, 5 min @72 °C and finally 4 °C. At the end of cycle 5, the PCR machine was paused, and 1.5 μl 10 μM A-PE-PCR10 was added (Table2 ), and the tubes replaced in the PCR machine for the program to continue for another 25 cycles. To visually verify that the yields were similar, 10 μl of the PCR reactions was loaded on an agarose gel, and the remaining 40 μl from each PCR were mixed with 40 μl Binding Buffer (GeneJET Gel Extraction Kit) and 20 μl Isopropanol, to be purified according to protocol and eluted in 40 μl elution buffer.
These 50 μl PCR reactions with low D6x primer concentration was run for the 5 first cycles of the following program: 2 min @98 °C, (10s @98 °C, 20s @50 °C, 2 min @ 72 °C) 31 cycles, 5 min @72 °C and finally 4 °C. At the end of cycle 5, the PCR machine was paused, and 1.5 μl 10 μM A-PE-PCR10 was added (Table