α amanitin

Embryos were glued on a coverslip after dechorionation. The embryos were dehydrated for 12–15 min, covered in Halocarbon oil 700 (Sigma), and injected with α-amanitin (100mM in water, Santa Cruz), Dextran-Alexa488 (1mg/mL; Life Technologies), or water. After injection, the embryos were placed on an air-permeable membrane and imaged on the spinning disk confocal microscope.

To deplete ATP, cells were treated with 6 mM 2-deoxyglucose (DOG) and 1 µM trifluoromethoxy-carbonylcyanide phenylhydrazone (FCCP) dissolved in CO₂-independent medium supplemented with L-glutamine 2 hr before imaging. For cytoskeletal perturbations 10 µM latrunculin A, 10 µM blebbistatin or 10 µM nocodazole, respectively, in CO₂-independent medium supplemented with L-glutamine were added to cells 30 min before imaging. For chromatin perturbations, 20 µg/mL α-amanitin (Santa Cruz Biotechnology), 5 µg/mL cycloheximide (Santa Cruz Biotechnology), 83 nM flavopiridol (Santa Cruz Biotechnology), or 624 nM trichostatin A (TSA), respectively, in CO₂-independent medium supplemented with L-glutamine were added to cells 30 min, 30 min, 2 hr, and 24 hr before imaging, respectively. For cycloheximide, additional timepoint, 6.5 hr after drug additon, was evaluated. All chemicals were from Sigma Aldrich unless stated otherwise.

For the in vivo stability assay, α-amanitin treatment of ESCs was performed. Briefly, WT, TKO, Tet2WT, and Tet2ΔRBD ESCs were treated with 10 μg/ml α-amanitin (Santa Cruz) or with an equal volume of vehicle (water) as a control for 0 or 4 h, respectively. For the rescue experiments, TKO ESCs were transfected with the Tet2FL or Tet2 catalytic mutant (Tet2Mut)^{30 (link)} plasmid with JetPrime polyplus reagent according to the manufacturer’s instructions. They were then treated with α-amanitin as described above. The cells were then collected after washing with PBS and processed for quantitative PCR analysis and/or RNA-Seq. For RNA-Seq, total RNA was extracted from α-amanitin-treated cells and untreated control cells and depleted of ribosomal RNA. The RNA in this fraction was fragmented before library preparation and deep sequencing, as described above. All primers used in this study are described in Supplementary Data 8.

Human embryonic kidney 293T and mouse NMuMG cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) media supplemented with 10% fetal bovine serum, penicillin and streptomycin at 37°C and 5% CO2. For the α-amanitin treatment experiment, HEK293T cells were transfected with SV40 promoter-driven firefly luciferase reporter (pGL2-pro), or a construct containing a copy of TCTCGCGAGA. 24h post-transfection, cells were treated with 5 μg/ml α-amanitin (Santa Cruz Biotechnologies) as described [27 (link)] or with PBS (control), and firefly and Renilla luciferase bioluminescence activities were measured 24h after treatment.