Tet o fuw ascl1

To generate DOX-inducible lentiviral vectors, LeGO-G2 (Addgene #25917), a third-generation lentiviral vector, was first digested with EcoRI and PspOMI (to excise SFFV promoter and GFP gene) and further ligated with Tet operator, which was obtained from plasmid Tet-O-FUW-Ascl1 (Addgene #27150) using PCR amplification, cloned into T-vector (Promega) and finally digested with EcoRI and PspOMI. The resulting plasmid was named LeGO-TRE. The polycistronic cassette carrying all four factors linked with 2A peptides was excised from Tet-O-FUW-OSKM (Addgene #20321; courtesy of Dr. Kiselev) and subsequently cloned into the EcoRI site of LeGO-TRE. The resulting vector was named LeGO-TRE-OSKM. To create DOX-inducible GFP vector (LeGO-TRE-GFP), fragment coding GFP was excised from pLeGO-G2 using BamHI and NheI and then ligated into BamHI and NheI linearized LeGO-TRE.
To generate the lentiviral vector carrying transactivator M2rtTA (for expression of factors from TRE-promoters), eGFP was excised from LeGO-G2 with EcoRI and BamHI and replaced (using the same enzymes) by M2rtTA gene derived from FUW-M2rtTA (Addgene #20342) using PCR. The resulting vector was named LeGO-M2rtTA.

pMXS-Oct4, pMXS-Sox2, pMXS-Klf4, pMXS-Myc, pLKO.1 and pWPXLd plasmids were purchased from Addgene. Short hairpin RNAs (shRNAs) against Trp53, FosL1, Atoh8 and Sfrp1 were designed using the MISSION shRNA library from Sigma–Aldrich and ligated using the Rapid DNA ligation kit (Sigma–Aldrich) into the pLKO.1 vector digested with AgeI and EcoRI. Supplementary Table 2 lists the shRNA sequences. Atoh8 and Bcl11b complementary DNA was amplified from MEFs and cloned into the pWPXLd expression vector at the BamH1 restriction site. For Atoh8 ChIP-Seq, AM-Tag was added at the carboxy terminal. Single guide RNA targeting Atoh8 (designed using the CRISPOR program) was cloned into the lentiCRISPRv2 plasmid at a BsmBI restriction site. The single guide RNA sequences are listed in Supplementary Table 2. The pWPIR H-ras G12V and cyclin E plasmids were kindly supplied by the laboratory of A. Puisieux. Tet-O-FUW-Brn2, Tet-O-FUW-Ascl1, Tet-O-FUW-Myt1l, FUW-TetO-Sall4, FUW-TetO-Nanog, FUW-TetO-Esrrb, FUW-TetO-Lin28 and FUdeltaGW-rtTA plasmids were purchased from Addgene.