Plasmids and Cloning
A sequence containing two BSMBI restriction sites flanked by hnRNPA1
recruiting loops (ARLs) was cloned into PCDNA 3.1+ (Invitrogen) behind
the CMV promotor between NheI and XbaI restriction sites, upstream of
the polyadenylation sequence to create a Golden Gate vector for ARO
constructs. This vector was then used to clone all additional ARO
constructs except for scrOligo-ARL, Oligo, ARL-Oligo, and Oligo-ARL
(Figure 2A) which were cloned into the same locus via Gibson assembly.
All Golden Gate cloned constructs were ordered as two single-strand DNA
oligos (Integrated DNA Technologies) and annealed such that overhangs
were compatible with the Golden Gate ARO vector. Once cloned, these
annealed oligos served as the targeting oligos for the AROs.
Single-strand RNA ARO oligos were ordered directly from IDT (Integrated
DNA Technologies) and contained two phosphorothioate bonds at each end
to protect the RNA from degradation by exonucleases. The sequence used
for the ARL was as follows: 5’- GGATCCATTCGTAGGAGGAACGGATCC-3’.
Sequences for targeting oligos in AROs used in experiments are as
follows. ARL-Oligo-KRT14 -ARL: 5’CCAGAGGAGAACTGGGAGGAGG-3’,
scrambledARL-Oligo: 5’-TGTGGCGAGTAGACTCGAAG-3’, ARL-TBK1-O1-ARL:
5’-GTTCCCTGAGAACTGGAAAG-3’, ARL-TBK1-O2-ARL: 5’-CCTGAAGACTGGTTTCTATT-3’,
ARL-TBK1-O3-ARL: 5’- TCTGCCAGTGATCCACCTGG-3’, ARL-TBK1-O4-ARL: 5’-
CTTCTTGATGTGCCCATGCG-3’, ARL-TBK1-O6-ARL: 5’- TCTGTCTTTCGGATGAGTGC-3’.