Lentiviral shRNA and Overexpression Constructs

shRNA oligo sequences were obtained from the RNAi Consortium (TRC, Broad Institute) and cloned into pLKO.1 vector (Addgene, #10878) according to a corresponding Addgene protocol (65 (link)). Four to five shRNAs per gene were tested for their knockdown potential and the two most efficient shRNAs were used in experiments. The non-targeting TRC2 shRNA (referred to as scrambled shRNA shScr, Sigma-Aldrich, #SCH202) was used as a negative control. For shRNA sequences see table S9. The Myc-Flag-tagged cDNA for mouse p21 was obtained from Origene (#MR227529) and subcloned into the lentiviral pTSIN-PGK-puro2 backbone (43 (link), 66 (link)) or dox-inducible pTRIPZ-PKG-puro backbone (modified from GE Dharmacon) (67 ). Similarly, the Myc-Flag-tagged cDNAs for mouse p16 (Origene, #MR227284) and mouse p27 (Origene, #MR201957) were also subcloned into the lentiviral pTSIN-PGK-puro2 backbone.

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Sturmlechner I., Zhang C., Sine C.C., van Deursen E.J., Jeganathan K.B., Hamada N., Grasic J., Friedman D., Stutchman J.T., Can I., Hamada M., Lim D.Y., Lee J.H., Ordog T., Laberge R.M., Shapiro V., Baker D.J., Li H, & van Deursen J.M. (2021). p21 produces a bioactive secretome that places stressed cells under immunosurveillance. Science (New York, N.Y.), 374(6567), eabb3420.

Publication 2021

pLKO.1 vector scrambled shRNA shScr

Backbone Cdnas Gene Mouse Oligo Rnai Shrna Vector

Corresponding Organization : Mayo Clinic in Arizona

Other organizations : University Medical Center Groningen, University of Groningen, WinnMed, Unity Biotechnology (United States)

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Variable analysis

independent variables

ShRNA oligo sequences obtained from the RNAi Consortium (TRC, Broad Institute) and cloned into pLKO.1 vector
Myc-Flag-tagged cDNA for mouse p21, p16, and p27 subcloned into lentiviral pTSIN-PGK-puro2 backbone or dox-inducible pTRIPZ-PKG-puro backbone

dependent variables

Knockdown potential of 4-5 shRNAs per gene

control variables

Non-targeting TRC2 shRNA (referred to as scrambled shRNA shScr, Sigma-Aldrich, #SCH202) used as a negative control

controls

Positive control: Not explicitly mentioned
Negative control: Non-targeting TRC2 shRNA (referred to as scrambled shRNA shScr, Sigma-Aldrich, #SCH202)

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