ExtData_Figure_2_replicates

(A) Xrn1 knock out A549 cells were infected with WT PR8 or PR8-PA(∆X), or mock infected. RNA was isolated, 5’ RACE was performed using primers specific for BCAP31, TUBA1B, INSIG1 or SLC7A5, and the PCR products were run on an agarose gel. Primers were positioned ~200-300 nucleotides downstream of the predicted cut sites.  

(C) HEK293T ishXrn1 cells were treated with no drug or doxycycline for 3-4 days to induce shRNAs against Xrn1, then protein lysates were collected and probed with antibodies against Xrn1, or b-tubulin as a loading control to check for efficient knock down of Xrn1.

(E) HEK293T ishXrn1 cells were treated with doxycycline for 3-4 days to induce knock down of Xrn1, then transfected with luciferase reporters containing 99 bp insertions from the indicated genes (INSIG1, SLC7A5, STOML2, YKT6, TUBA1B, and BCAP31), and where indicated, with PR8 PA-X. RNA was extracted and used to run 5’ RACE. For all gels, the DNA bands were purified and sequenced to confirm their identities. The SLC7A5 sequence was only consistently cut when inside the luciferase reporter.

R# --> replicate number; LG### --> experiment number.

.sgd files are the original source files acquired with the Syngene Imager.