Kod mutagenesis kit

The gene encoding TERT was amplified from the plasmid pCDH‐3xFLAG‐TERT, which was a gift from Steven Artandi at Stanford Cancer Institute Core, Stanford University (Addgene plasmid #51631; http://n2t.net/addgene:51631). The mVenus sequence was isolated from pCS2‐mVenus plasmid, which was purchased from the RIKEN BioResorce Research Center (Ibaraki, Japan, RDB15116). pSBbi‐Pur was a gift from Eric Kowarz at Institute of Pharmaceutical Biology, Goethe‐University (Addgene plasmid #60523; http://n2t.net/addgene:60523). The gene encoding TERT and mVenus was inserted into pSBbi vector using the In‐Fusion cloning kit (Takara Bio, Shiga, Japan). The R3E/R6E mutation was introduced using the KOD mutagenesis kit (Toyobo, Osaka, Japan). The Y707F mutation was introduced by PCR. All plasmids were transformed into DH5α chemical competent cells and purified using the FastGene miniprep kit and endotoxin‐free miniprep kit (Nippon Genetics, Tokyo, Japan). The primer sets used are presented in Table S1.

The MISSION shRNA TRC1 vectors (Supplementary Table 2) used in this study were purchased from Sigma-Aldrich (MO, USA). The sequence of shPOGZ^miR30 (Supplementary Table 3) was designed using an online design tool (http://cancan.cshl.edu/RNAi_central/RNAi.cgi?type = shRNA). The template oligonucleotide carrying the shPOGZ^miR30 sequence was obtained from Eurofins Genomics (Tokyo, Japan), amplified via PCR and subcloned into a pCAG-miR30 vector. In order to generate a plasmid expressing mPOGZ, mPOGZ cDNA (DNAFORM, Kanagawa, Japan, clone ID: 30745658) was amplified via PCR and subcloned into a pcDNA-6Myc vector. Plasmids expressing mPOGZ mutants were generated using a KOD mutagenesis kit (Toyobo, Osaka, Japan) according to the manufacturer’s protocol.

Site-directed mutagenesis was performed with the KOD Mutagenesis kit (Toyobo, Osaka, Japan) as described42 (link). Then, the ligated PCR products were used for transformation of DH5α strain. The mutation in cDNA was verified before protein expression.

Three steps were needed for pUAST-gRNA construction. First, a fragment containing 3×P3RFP and the tubulin 3′ UTR was amplified from the pM3×P3-RFPattP′ vector (Bischof et al. 2007 (link)) and used to replace the white gene between two EcoRV sites in pUAST; this created the entry plasmid pUAST-RFP. Second, a 400-bp Drosophila U6B promoter sequence (Wakiyama et al. 2005 (link)) and the 1133-bp promoter sequence from Drosophila non-coding RNA CR34335 (Graveley et al. 2010 (link)) (Figure S4) were separately cloned into the pMD19-TA gRNA scaffold vector (stock in our laboratory) to create TA-U6B-gRNA and TA-CR7T-gRNA, respectively. The KOD mutagenesis kit (TOYOBO, Japan) was used to insert 20-bp target sequences between the promoter and the gRNA scaffold. Third, the target-specific U6B/CR7T-gRNAs were released by SpeI/KpnI and cloned using the same sites in pUAST-RFP, generating the final transgenic gene-specific vectors (Figure S6). In total, five transgenic gRNA vectors were generated: pUAST-U6B-k81-gRNA for targeting the ms(3)k81 locus; pUAST-U6B-y1-gRNA and pUAST-CR7T-y2-gRNA for targeting the yellow locus; and pUAST-CR7T-w1-gRNA and pUAST-CR7T-w2-gRNA for targeting the white locus. All primers are listed in Table S2 and Table S3.

For gain-of-function experiments, we used the pMXs retroviral expression system as previously described [2 (link)]. Retroviral vectors expressing deletion mutant of NFIA were constructed using the KOD mutagenesis kit (TOYOBO) according to the manufacturer’s instructions. A retroviral vector expressing Cre recombinase was a gift from Kai Ge (Addgene plasmid # 34564).

Full-length human METTL23 cDNA was cloned into the pEF-BOS-FLAG vector (20 (link)) using the standard procedure. To introduce mutations into expressing vectors, splicing deletion products were obtained using the KOD Mutagenesis Kit (TOYOBO, Japan) and specific deletion primers (Supplemental Table 7) and were then validated by Sanger sequencing.

ACC1 or Pin1 plasmids were prepared, as follows. Briefly, cDNAs encoding human ACC1 or human Pin1 with S -tag at their N terminal, were inserted into each Flag-pcDNA or pcDNA3.1 (-). Mutants with one substitution were created by using a KOD mutagenesis kit (TOYOBO).