GSM9679991: KOLF_KO_rna_rep2; Homo sapiens; RNA-Seq
Control or METTL5-KO NPCs were grown on 15cm plates (3 plates were combined to make 1 replicate, and control and KO had three biological replicates each) and harvested at 80% confluency and immediately treated with cycloheximide (100 µg/mL) for 3 minutes at 37°C to halt ribosomes on mRNAs. Cells were washed once with ice-cold PBS containing cycloheximide (100 µg/mL) and lysed in polysome lysis buffer [20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl₂, 1% Triton X-100, 1 mM DTT, 100 µg/mL cycloheximide, DNAse I 25U/ml), H20], followed by a 30 minute incubation on ice. Lysates were clarified by centrifugation at 10,000 X g for 10 minutes at 4°C. 1-2ug of RNA was saved for RNA-seq. 30ug of RNA was incubated with 30U/ml RNAse P1 for 1hr at 37°C. The Illustra MicroSpin Columns S-400HR were used for monosome isolation and downstream steps were the same as reported in McGlincy & Ingolia, 2017. RNA-seq:1-2 ug of RNA was purified using the Direct-zol RNA miniprep kit (Zymo) then used in RNA seq library prep kits: NEBNext rRNA Depletion Kit V2 (human/mouse/rat) (NEB), NEBNext Ultra II Directional RNA Library Prep Kit of Illumina (E7760), with NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) (E6440S) Ribo-seq: library was constructed in the same manner as McGlincy & Ingolia, 2017.