Extended Data Figure 5: Effects of CAF-1 dose on NIH3T3 growth and reprogramming potential.

a, Competitive proliferation assay to determine effect of indicated Chaf1a and Chaf1b shRNA vectors on long-term growth potential of immortalized NIH3T3 cell line. Cells were infected with indicated constructs and the fraction of shRNA vector-positive cells was measured by flow cytometry at different time points. Data were normalized to cell counts at day 2 post-transduction. Rpa3.455, validated control shRNA targeting the broadly essential replication protein A3. Error bars show s.d. from biological triplicates. b, Histogram plots of MEFs harbouring R26-M2rtTA allele and either Col1a1::tetOP-miR30-tRFP-Ren.713 or Col1a1::tetOP-miR30-tRFP-Chaf1a.164 shRNA knock-in allele after transduction with pHAGE (Ef1a-OKSM) lentiviral vector and exposure of cells to different doses of doxycycline for 2, 4 and 6 days. Low doses of doxycycline (0.2 µg ml⁻¹) result in lower expression of the shRNA miR cassettes than high doses of doxycycline (2 µg ml⁻¹). c, Quantification of data shown in b using the geometric mean (n = 1 experiment for 3 indicated time points). d, Reprogramming efficiency of Col1a1::tetOP-miR30-tRFP-Chaf1a.164; R26-M2rtTA MEFs infected with pHAGE (Ef1a-OKSM) vector and induced with high (2 μg ml⁻¹) or low (0.2 μg ml⁻¹) doses of doxycycline for indicated number of days before scoring for Nanog⁺ iPS cells by immunocytochemistry on day 9. e, Classification of CRISPR/Cas9-induced mutations by sequence analysis of representative iPS cell clones (wt, wild type; indel, insertion/deletion; fs, frameshift; *, point mutation). f, Western blot analysis for CAF-1 subunits p150 and p60 in 6 representative iPS cell clones after CRISPR/Cas9-induced modifications of the Chaf1a locus (see Supplementary Fig. 1 for full scans). Wt/wt samples show unmodified wild-type control samples.