Pog44 plasmid

Promoter transgenes were integrated into the 5A5 Flp-In cell clone by co-transfecting the reporter constructs with the pOG44 plasmid (Invitrogen) encoding a modified yeast Flp enzyme with optimal stability at 37 °C and reduced recombinase activity. We determined that 9:1 mass ratio of pOG44 to the transgene reporter vector gave optimal number of cell clones. The pcDNA5/CAT/FRT plasmid (Invitrogen) was co-transfected with pOG44 into the 5A5 cell clone as a positive control. Transfection was performed in six-well plates. Twenty-four hours after transfection, cell culture media was replaced with growth media without antibiotics. Forty-eight hours after transfection, transfected cells from each well were trypsinized and resuspended in the selection media (growth media +1 mg/ml Hygromycin). We changed the selection media every three or four days. Cell foci became visible usually 8–14 days after plating. We picked individual cell clones by 8-mm diameter cloning cylinder (Fisher Scientific) and resuspended them into 24-well plates. Cell clones were expanded into 10-cm plates. We then froze cells and isolated genomic DNA (DNeasy kit, Qiagen) and total RNA (RNeasy Plus kit, Qiagen) for analysis.

DNA sequences for the HTT_ex1-GFP constructs were taken from the yeast expression plasmids and inserted into the pcDNA5-FRT-TO (Thermo Fisher Scientific) mammalian expression vector allowing doxycycline-inducible expression of the construct. At 80%–90% confluency, cells were transfected with expression plasmids using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer’s protocol, and HTT_ex1-GFP expression was induced using doxycycline (150 ng/mL). The Flp-In HEK293 HTT_ex1-GFP (25 and 97QP) isogenic stable cell lines were generated using Flp-In System (Invitrogen) following the manufacturer’s protocol. Briefly, pcDNA5-FRT-TO expressing HTT_ex1-GFP constructs were co-transfected with pOG44 plasmid (Invitrogen), which constitutively expresses the Flp recombinase. Stable Flp-In expression cell lines were selected for hygromycin resistance. HTT_ex1-GFP was induced using doxycycline (150ng/mL).

All HeLa cell lines used in this study (Supplementary Data 1) were generated from a Flp-In T-REx HeLa cell line created by Stephen Taylor and colleagues^{61 (link)}. The Flp-In T-REx HeLa cells were transfected with the pcDNA5/FRT/TO derived plasmids and the pOG44 plasmid (Thermo Fisher Scientific) according to the manufacture’s protocol. Stable cell lines were generated by isolation of single colonies, which were viable in the DMEM medium containing 10% tetracycline-free FBS, 2 mM l-glutamine, 250 µg mL⁻¹ hygromycin and 4 µg mL⁻¹ blasticidin. Protein expression was verified using western blotting (uncropped blot images are included in a Source Data file) and the following antibodies were used: anti-HJURP antibody (Abcam, ab100800; dilution, 1:500), anti-Mis18α antibody (Thermo Fisher Scientific, PA5-53771; dilution, 1:500), anti-SNAP-tag antibody (NEB, P9310; dilution, 1:1,000), anti-α-tubulin antibody (Sigma, T9026; dilution, 1:8,000), anti-vinculin antibody (Sigma, V9131; dilution, 1:10,000), anti-mouse HRP-conjugated antibody (GE, NXA931-1ML; dilution, 1:10,000), anti-rabbit HRP-conjugated antibody (GE, NA934-1ML; dilution, 1:10,000).

To generate stable cell lines expressing 3xFLAG-tagged ALKBH1, TET2, and MPP8, Flp-In TRex 293 cells were seeded at 0.6 × 10⁶ cells per well in six-well plates, and co-transfected with the appropriate pCDNA5/FRT/TO-3xFLAG-tagged plasmid (0.2 µg) and pOG44 plasmid (2 µg, Thermo Fisher). After selection with 100 µg/mL hygromycin B and 15 µg/mL blasticidin, colonies were expanded. To confirm expression of the desired protein, cells were induced with tetracycline (0–1 µg/mL) for 24 h. Cells were harvested and lysed in NP-40 lysis buffer [50 mM Tris HCl pH 7.5, 150 mM NaCl, 5 mM MgCl₂, 0.5% NP-40, 1 mM PMSF (supplemented freshly), protease inhibitor tablet (Roche, supplemented freshly)]. Protein concentration was normalized to 2 mg/mL, and proteins were separated by SDS-PAGE and analyzed by western blot with anti-FLAG M2 antibody (Sigma, #F1804, 1 µg/mL, 1:1000).