Rnatri

Naïve and memory CD8+ T cells were generated using the OT-I system as described previously (32 (link),33 (link)). RNA-Seq was done essentially as described (34 (link)). Briefly, total RNAs were prepared using RNA-Tri (Bio&SELL) and further purified using the RNeasy mini kit (Qiagen) in combination with a DNase I (Qiagen) treatment. RNA sequencing libraries were prepared by using the TruSeq mRNA Library Preparation kit (Illumina). 125-bp paired-end reads were generated by using a HiSeq 2500 sequencer (Illumina) with V4 sequencing chemistry. Triplicate samples from naïve and memory T cells were sequenced (around 40 × 10⁶ reads per sample) and analyzed using a MISO-based pipeline (35 (link)).

RNA was extracted from ∼1 × 10⁶ cells using RNATri (Bio&Sell) and 0.5 μg of total RNA was used for reverse transcription with the UPF1 reverse primer (sequence given below). To detect the mRNA corresponding to the two isoforms of human UPF1, we designed primers specific to sequences in exons 7 and 8, which flank the alternative 5′-splice site and, upon amplification, would generate products of 166 and 133 bp for human UPF1₁ and UPF1₂ isoforms, respectively. The primer sequences are as follows: forward 5′-GGGACCTGGGCCTTAACAAGAAGAGA-3′ and reverse 5′-ATCCCTTTCCACAGGGGCGCAAGGT-3′. Plasmids expressing the human UPF1₁ΔCH and UPF1₂ΔCH proteins were used as positive controls. For quantification of mRNA levels of the two isoforms low-cycle PCRs were performed with the ³²P-labeled forward primer (sequence given above), products were resolved by denaturing-PAGE and analyzed using a Phosphoimager and the ImageQuantTL software (GE Life Sciences). The mRNA preparations of different mouse tissues were analyzed as described above, using the human-specific forward primer (sequence given above, identical to the mouse sequence in this region) and the following mouse-specific reverse primer: 5′-ATCCCCTTCCACAGGGGCGCCAGAT-3′.

Total RNAs from control siRNA and U2AF35 siRNA transfected cells, as well as Q157R, Q157Rdel, and Q157P rescue cell lines, were purified using RNA-Tri (Bio&SELL) and further purified using the RNeasy mini kit (Qiagen) in combination with a DNase I (Qiagen) treatment to prevent genomic DNA contamination. RNA sequencing libraries were prepared by using the TruSeq mRNA Library Preparation kit (Illumina). Of note, 125-bp paired-end reads were generated by using a HiSeq 2500 sequencer (Illumina) with V4 sequencing chemistry. Biological duplicates were sequenced for all conditions.

RT–PCRs were done as previously described (Preussner et al., 2014). Briefly, RNA was extracted using RNATri (Bio&Sell) and 1µg RNA was used in a gene‐specific RT reaction. For analysis of minigene splicing, the RNA was additionally digested with DNase I and re‐purified. Low‐cycle PCR with a ³²P‐labeled forward primer was performed; products were separated by denaturing PAGE and quantified using a Phosphoimager and ImageQuantTL software. For qRT–PCR, up to four gene‐specific primers were combined in one RT reaction. qPCR was then performed in a 96‐well format using the ABsolute QPCR SYBR Green Mix (Thermo Fisher) on Stratagene Mx3000P instruments. qPCRs were performed in duplicates, mean values were used to normalize expression to a housekeeping gene (mouse: Hprt, human: Gapdh, plants: Ipp2; ΔCT), and Δ(ΔCT)s were calculated for different conditions. See Table EV2 for primer sequences.