title: Barcode RNA-Seq MPRA, Total sample, pTRP1-based, MIX3, A. thaliana, repeat 1
design: The protocol was adapted from (Szabo et al. 2020) and Click-iT Nascent RNA Capture Kit from Invitrogen manual. Arabidopsis protoplasts were transformed with MIX3 following the same procedure as for the MPRA in Arabidopsis protoplasts. After transformation, the samples were incubated for 5h and 40 minutes at 25C under constant light (85 mol/m2/s). 200 mM 5-ethynyl uridine (5-EU) stock solution (Click-iT Nascent RNA Capture Kit, Invitrogen, C10365) was added to each sample to a final concentration of 200 M and the samples were incubated for additional 20 minutes before harvesting. Total RNA was extracted from the frozen pellets as described above. DNase I treatment was repeated three times for each sample. mRNA was isolated from the total RNA as described above. In the elution step, the beads were resuspended in 55 L 10 mM TrisHCl (pH 7.5) and then re-eluted with the first eluate. 5-8 L of mRNA was set aside for preparation of libraries from total mRNA, whereas the remaining mRNA was split in three aliquots and each aliquot was used for one Click reaction with 0.25 mM biotin azide following the Click-iT kit manual. Biotinylated mRNA was precipitated from each Click reaction following the manual and resuspended in 25 L RNase-free water. 3 L bead suspension was added to each aliquot of biotinylated mRNA and incubated for 30 minutes at room temperature rotating at 30 rpm. After incubation, the three aliquots were pooled and washed five times with wash buffer 1 and five times with wash buffer 2. The bead suspension was resuspended in 50 L wash buffer 2 and used immediately for cDNA synthesis. Libraries were prepared from the total mRNA - mRNA samples were diluted to 50 uL final volume with RNase-free water. From each sample, four reactions with 11 L mRNA each and a construct-specific primer were prepared for cDNA synthesis using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, 18090010). RNase A treatment and purification with AMPure XP beads was performed. In the final step, the RT reactions were eluted in 73 L 10 mM TrisHCl (pH 8) buffer. The purified cDNA from the RT reactions was used for preparation of three PCR reactions amplifying the pTRP1-based transcripts. PCR reactions were incubated for 24 cycles. A second purification with AMPure XP beads was performed. Finally, all samples were analyzed with 5200 Fragment Analyzer (Agilent, M5310AA) and sequenced on an Illumina NovaSeq 6000.