Topo ta kit

Founder animals were identified by PCR using primers flanking the target loci that amplified a 601-bp fragment in wild-type animals (Rnbglap-genF1, 5′-GGCTCAGGCAGTGGATATAAA-3′; Rnbglap-genR1, 5′-CACAACTCCTCCCTACCAATATG-3′). Positive samples were confirmed by modified Sanger sequencing. Heteroduplex formation of amplified fragments was facilitated by denaturing the PCR samples at 95°C for 10 min and slowly cooling the samples to 4°C over ∼20 min to enable renaturation. The re-annealed samples, which included homoduplexes and heteroduplexes, were run on 6% polyacrylamide-TBE gels at 100 V for 45 min before staining with ethidium bromide to visualize bands under UV light. Samples showing heteroduplex mobility shifts were cloned into a plasmid vector (pCR 2.1) using the TOPO-TA kit (Thermo Scientific, Waltham, MA). Recombinant plasmids with inserts were isolated and subjected to Sanger sequencing to obtain sequence information of modified alleles.

A haemaglutinin (HA) tag flanked by XbaI and BamHI sites was cloned into the N-terminal of YBX1 cDNA of a pc3-DNA backbone using a pcDNA3.3-TOPO cloning kit (Thermo Fisher Scientific). Serine to either alanine or aspartic acid mutations at specific serine residues (S102, S165, S176) were generated in the pcDNA3.3 HA-YBX1 plasmid by site-directed mutagenesis using mutagenesis primers (Table S8) and were subsequently amplified with a Kapa high fidelity polymerase (Kappa HiFi PCR kit #KK2502, Roche Diagnostics, Rotkreuz, Switzerland). To introduce a FLAG tag to the 3’ end of HA-YBX1, two unique restriction sites, an EcoR1 site near the 3’ end of YBX1 and an Xho1 site, were used for cloning in conjunction with the TOPO-TA kit (Thermo Fisher Scientific). The DNA removed by these nucleases was replaced with synthetic oligonucleotides. The oligonucleotides contained compatible ends to replace the 3’ end of YBX1 with the original sequence while incorporating an in-frame FLAG tag before the termination codon.

To ensure that the accumulation of NSUN4 on the mtLSU in GTPBP6-deficient cells does not affect its second function as a methyltransferase modifying the 12S rRNA at position 1488, we analyzed the modification status of 12S-m⁵C1488 and, as a control, 12S-m⁴C1486, by subjecting DNase-treated total RNA from HEK293T wild-type and Gtpbp6^−/− cells to bisulfite sequencing^{50 (link),51 (link)}. Bisulfite treatment was performed using the EpiTect Bisulfite kit (Qiagen) according to the manufacturer’s instructions. Deamination was performed by three cycles of incubation at 70 °C for 5 min and at 60 °C for 60 min. Samples were purified using mini Quick spin RNA columns (Roche) and the desulphonated in Tris pH 9.0 for 30 min at 37 °C. RNA was extracted using phenol:chloroform, precipitated and reverse transcribed from the 12S-m⁵C841_RT primer (Primer sequences: Supplementary Table 4) using Superscript III reverse transcriptase (Thermo) according to the manufacturer’s instructions. A 70-nt fragment of the 12S rRNA was amplified by PCR (Primer sequences: Supplementary Table 4) and cloned using a TOPO-TA kit (Thermo). Clones were sequenced at Eurofins Genomics using the T7 primer and only sequences in which all cytosines (disregarding C1486 and C1488) were converted to uracil/thymine were used for the presented analysis (Supplementary Fig. 6c).