2P-seq of nuclear RNA with modifications

2P-seq was performed as described before (Spies et al., 2013 (link)) with several modifications. First, nuclear RNA was used instead of total RNA. Second, the primer sequence was re-designed (Supplementary file 2), so that multiple libraries could be sequenced in the same lane using Illumina sequencer. Third, a lower concentration of RT primer was used during reverse transcription to reduce internal priming (Scotto-Lavino et al., 2006 (link)). Briefly, poly(A) RNA was purified using oligo-dT25 beads (Invitrogen) and eluted directly into 25 μl of RNase T1 buffer. RNase T1 digestion was performed for 20 min at 22°C using 0.5 U RNase T1 (biochemistry grade, Ambion). After this partial RNase T1 digestion, reverse transcription was performed by addition of 1 μl 1 μM RT primer. Single-stranded cDNAs of 200–400 nucleotides were purified on a 6% TBE-urea gel, then circularized by CircLigaseII (Epicentre). Circularized cDNA was amplified by high-fidelity PCR for 10 cycles using barcoded PCR primers, then gel purified and sequenced using the Illumina Read one primer.