Barcode RNA-Seq MPRA, pTRP1-based, MIX1, N. benthamiana, repeat 3
The protocol was adapted from Neumayr et al.(35) and Jores et al.(26). From each replicate, ten reactions with 11 L mRNA each and a construct-specific primer (P37-P40) were prepared for cDNA synthesis using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, 18090010). One of the reactions was used as no reverse transcription control, in which the RT enzyme was replaced with RNAse-free water. After the cDNA synthesis, 1 L RNase A (200 g/mL) was added to each reaction and the samples were incubated at 37C for 1h. The nine RT reactions were pooled and purified with 1.8 volumes of AMPure XP beads (Beckman Coulter, A63880) to each volume of cDNA. The no RT control was processed separately in the same way as the RT reactions. In the final step, the RT reactions and the no RT control were eluted in 146 L and 49 L 10 mM TrisHCl (pH 8) buffer, respectively. The purified cDNA was split in two 72 L aliquots and each aliquot was used for preparation of three PCR reactions amplifying the 35S-based transcripts (P17-20 + P41) and pTRP1-based transcripts (P33-36 + P41), respectively. The no RT control was also split in two 24 L aliquots and each aliquot was used for one PCR reaction of the 35S and pTRP1 transcripts, respectively. All PCR reactions were prepared with 24 L template, 25 L KAPA HiFi HotStart ReadyMix (Roche Molecular Systems, KK2601), 0.5 L forward primer (100 M) and 0.5 L reverse primer (100 M). The samples from N. benthamiana tissue were amplified with 21 cycles, whereas the ones from A. thaliana, tomato and maize protoplasts - with 24 cycles. The three reactions from each library (35S and pTRP1) were pooled and purified with an equal volume of AMPure XP beads. The two no RT reactions (35S and pTRP1) were processed separately in the same way. Finally, all samples were analyzed with 5200 Fragment Analyzer (Agilent, M5310AA) and sequenced on an Illumina NovaSeq 6000 or NextSeq 2000.