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nature research | reporting summary
April 2018CD8a(cat. 100704, clone 5.3-6.7, lot. B257790), biotin anti-mouse CD3e(cat. 100304, clone 145-2C11, lot. B268248), biotin anti-
mouse B220(cat. 103204, clone RA3-6B2, lot. B268525), biotin anti-mouse Ly60G/Ly6C (Gr-1) (cat. 108404, clone RB6-8C5, lot.
B236916), biotin anti-mouse TER119(cat. 116204, clone TER-119, lot. B256623), FITC anti-mouse CD8a (cat. 100706, clone
53-6.7, lot. B259672), PE/Cy7 anti-mouse CD16/32(cat. 101318, clone 93, lot. B249751), PE/Cy7 anti-mouse TER119(cat. 116222,
clone TER-119, lot. B251241), APC anti-mouse Ly6G/Ly6C(Gr-1) (cat. 108412, clone RB6-8C5 ), APC anti-mouse TER119(cat.
116212, clone TER-119, lot. B246740), APC anti-mouse CD117(c-Kit) (cat. 135108, clone ACK2, lot. B255217), APC/Cy7
Streptavidin (cat. 405208, lot. B258702), Brilliant Violet 510 anti-mouse CD41 (cat. 133923. clone MWReg30, lot. B258166),
Brilliant Violet 510 anti-mouse CD71 (cat. 113823, clone RI7217, lot. B301604) and Pacific Blue anti-mouse Ly-6A/E(Sca-1) (cat.
108120, clone D7, lot. B258720) were purchased from BioLegend. APC anti-mouse CD44(cat. 17-0441-82, clone IM7, lot.
4312474), and PE-Cyanines5 anti-Hu/Mo CD45R(B220)(cat. 15-0452-83, clone RA3-6B2, lot. 2052744) were purchased from
eBioScience. FITC rat anti-mouse CD25(cat. 1595-02S, lot. 17103-P624AF), Goat anti-mouse IgM (cat. 1020-09, lot. K2915-5357)
were purchased from Southern Biotech). PE rat anti-mouse CD4(cat. 557308, clone GK1.5, lot. 8093955), PE rat anti-mouse
CD19 (cat. 557399, clone 1D3, lot. 7213714) and biotin rat anti-mouse CD5(cat. 553018, clone 53-7.3, lot 8115630), FITC anti-
mouse CD43(cat. 553270, clone S7, lot. 6110908), and Alexa Fluor 700 rat anti-mouse CD34(cat. 560518, clone RAM34, lot.
8116836) were purchased from BD Pharmingen. All FACS antibodies were used at 1:200 dilution. Antibodies used for
Immunofluorescence: Mouse anti-Hu/Mo Ku80(111) monoclonal antibody (MA5-12933) was purchased from ThermoFisher and
used at 1:100 dilution. Rabbit anti DDX21 polychonal antibody (NB100-1718) was purchased from Novus and used at 1:500
dilution. Mouse anti-DNA-PKcs Ab-4 (Cocktail) monoclonal antibody (MS423P) was purchased from ThermoFisher and used at
1:100 dilution. Alexa Fluor 488 conjugated goat anti-Rabbit secondary antibody (cat. A-11008, lot. 1937184), Cyanine3
conjugated goat anti-mouse secondary antibody (cat. A10521, lot. 1126597), and Alexa Fluor 594 conjugated goat anti-Rabbit
secondary antibody (cat. A-11012, lot. 1745478) were purchased from Invitrogen and used at 1:500 dilution.Validation All antibodies are extensively used and well accepted commercial antibodies and validated in several published manuscript.
Antibodies were further validated in our lab by western blotting appropriate cell lysates and assessing the resulting western blot
for signal where the band of the expected molecular weight of the target protein would constitute at least 80% of the total signal
of that lane. In some cases we performed IP mass spec to further validate the specificity of the antibody. FACS antibodies were
validated by single staining on positive population from WT mouse, and Immunofluorescence antibodies were validated by
either comparative staining of WT cell line to corresponding knock out cell line, or by inhibitor treatment, for intensity and
position of positive signal. See methods for details. Additionally, our in vitro kinase assay with in vitro purified humans proteins
further validate the specificity of our DNA-PK, Ku86 and p53 antibodies.Eukaryotic cell lines
Policy information about cell lines
Cell line source(s) HeLa and IMR90 were purchased from ATCC and Sigma, respectively. U2OS cells were purchased from ATCC. Most MEFs, ES
cells and v-abl kinase transformed B cells were freshly derived from genetically modified animal models in the Zha lab, unless
noted below. Standard protocols (can by found in references) were used to derived those cell lines. Their genotypes were
confirmed and validated via PCR. More than 2, often more than 3 independently derived cell lines of each genotypes were
used for each measurement. A few control cells were generously provided by colleagues. Specifically, the DNA-PKcs-/-
Abeslon lines and the KU70-/- ES cells were originally derived in the laboratory of Dr. Frederick Alt at the Children's Hospital
at Boston. The DNA-PKcs3A/3A Abelson lines were generously provided by Dr. Barry Sleckman then at Cornell University and
now at the University of Alabama. Fand2-/- MEFs were generously provided by Dr. Agata Smogorzewska at the Rockefeller
University. The genotype of all these cell lines have been independently verified by PCR in the Zha lab to the best of our
ability.Authentication Commercial available cell lines were authenticated by the vendor, we have not re-authenticated the cells since we bought
them. Investigator derived cell lines were validated by PCR genotyping to the best of our ability.Mycoplasma contamination All cell lines are mycoplasma free. We routinely test for mycoplasma contamination.Commonly misidentified lines
(See ICLAC register)None of the cell lines used in this study are in the database of commonly misidentified cell lines.Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research
Laboratory animals This study includes experiments conducted on house mice (Mus musculus) of either pure 129S1/SvImJ strain, or 129S1/SvImJ
and C57BL/6J mixed strain, and of both male and female. No gender bias were noted. All analysis, unless otherwise noted (e.g.,
tumor and survivial cohorts), was conducted on mice of about 2 weeks old, or E14.5 embryos.Wild animals The study did not involve wild animalsField-collected samples The study did not involve samples collected from the field