HCT116 cells were maintained in DMEM with 10% fetal calf serum. Transfections were with JetPrime (polyplus). For CRISPR, 1 µg of guide RNA plasmid and 1 µg of each repair plasmid were transfected into six-well dishes. Twenty-four hours later, culture medium was changed, and, a further 24 h later, cells were split into a 100-mm dish containing 800 µg/mL neomycin and 150 µg/mL hygromycin. After ∼10 d of selection, single colonies were transferred to a 24-well plate and screened by PCR or Western blotting. The presence of repair cassettes at XRN2 or CPSF73 was confirmed by Sanger sequencing. An optimized sleeping beauty transposon system (Kowarz et al. 2015 (link)) was used to generate Tir1-expressing parental cell lines and cells in which Xrn2 derivatives were stably transfected. A 24-well dish was transfected with 300 ng of sleeping beauty plasmid (derived from pSBbi-puro/pSBbi-blast)) and 100 ng of pCMV(CAT)T7-SB100. Twenty-four hours later, cells were put under selection with 1 µg/mL puromycin or 20 µg/mL blasticidin. For Tir1-expressing cells, single colonies were isolated; for Xrn2 rescue experiments, the entire population was studied. Auxin (Sigma) was added to 500 nM for 60 min unless stated otherwise. TMP (Sigma) was maintained at 20 µM, and, for depletion, cells were grown in medium lacking TMP for 10 h unless stated otherwise. Act D and flavopiridol were used at 5 μg/mL and 1 μM, respectively.
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