Micrornaeasy kit

MOLM13 cells were transduced with an Empty or a METTL1 gRNA as described above. On day 5 post-transduction the cells were suspended in methionine-free RPMI-1640 media (Gibco) supplemented with 10% v/v FBS and 1% v/v penicillin/streptomycin/l-glutamine at a density of 1 000 000 cells mL–1. The cells were incubated for 30 min at 37 °C followed by addition of PropSeMet at a final concentration of 150 μM. Treated cells were incubated for further 16 h at 37 °C. Aqueous solutions of premixed CuSO4 and THPTA were added at final concentrations of 100 and 300 μM, respectively, followed by the click-degrader at 400 μM and NaAsc at 5 mM. Treated cells were incubated for 10 min at 37 °C and resuspended in complete RPMI-1640 medium. Afterward, the cells were again incubated at 37 °C and harvested after 5 h for RNA extraction. Total RNA was extracted from pelleted cells using microRNAeasy Kit (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was retrotranscribed using Superscript Vilo Master Mix (Invitrogen) according the to manufacturer’s instructions. Levels of specific RNAs were measured using fast mode of StepOnePlus Real-Time PCR System (Applied Biosystems) and Fast SYBR Green Master Mix (Applied Biosystems) according to the manufacturer’s instructions. RNA levels were normalized to 18S subunit of the ribosome. Primer sequences are listed in Table S8 .

iPSCs were grown on MEFs or Matrigel to confluence. iPSCs were dissociated with Accutase (Invitrogen, A11105-01). 3,000–6,000 single cells were placed in Aggrewell 400 plates (Stemcell Technologies, 27945) and incubated in hES media without FGF for 24 hours. Aggregates were released and passed through the 40 µm cell strainer to remove single cells. 1,000 aggregates/well were plated in an ultra-low adherence 6-well plate (Corning, 3471) in Neural Induction medium on day 0. Neuralized embryoid bodies (nEBs) were fed every other day and harvested on days one and eleven for further analysis. RNA was isolated from EBs using the MicroRNAeasy kit (Qiagen, 217004).

2 × 10⁴ sorted cells were mixed with loading dye and electrophoresed through 4-12% SDS-PAGE gels for Western blots. Total RNA was extracted with microRNA easy kit (Qiagen) and digested with DNase (Qiagen). Concentration was measured by Bio-analyzer (Agilent). Reverse transcription and real time PCR were performed using standard protocols. Antibodies, qPCR primer sequences, and a more detailed Western Blot protocol are included in the Supplemental Experimental Procedures.

Plants were grown on standard plates in standard conditions and transferred to CNQX or mock cutting board plates at 7 days after plating. Immediately after transfer, plants were cut at 270 µm (high cuts) and then transferred to CNQX plates or mock plates. Approximately 20 stump tips consisting of about 100 µm of the stump were collected at each time point (30 min, 4 h, 14 h, 24 h). For controls, two separate sets of uncut roots were transferred to CNQX plates or mock plates and incubated for 14 h before collecting root tip tissue, which also consisted of about 100 µm of the distal root tip. RNA was extracted using the Qiagen MicroRNAeasy Kit. We performed cDNA construction and library amplification with Nugen OvationRNA Amplification System V2. Libraries were made using the Nugen Ovation Ultralow DR Multiplex System (Tecan). DNA fragmenting was performed using the Covaris S220. Short read sequences were generated on an Illumina NextSeq 500 and were aligned using HISAT2. Counts were normalized between samples using DESeq2’s median of ratios method before analyzing for differentially expressed genes.