GSM2602073: MOLM13 METTL3 shRNA #9 RNAseq rep 1; Homo sapiens; RNA-Seq
miCLIP RNA extract protocol: Total RNA was extracted from MOLM13 cells with Trizol (n=3 biological replicates).  Poly(A) mRNA was then isolated using two rounds of selection with oligo(dT) magnetic beads (New England Biolabs).  30 ug of poly(A) RNA was used for each replicate in this study. miCLIP library construction protocol: miCLIP libraries were prepared as previously described (PMID: 28349454). RNA-seq and Ribo-seq library construction protocol: MOLM13 cells were transduced with control shRNA or two independent shRNAs targeting METTL3.  Four days post-transduction 3x10^6 MOLM13 cells were washed twice with ice-cold PBS pelleted by centrifugation.  After pelleting, cells were resuspended in 300 μl lysis buffer (25 mM HEPES-KOH pH 7.2, 200 mM KOAc, 1% NP-40, 10 mM MgCl2, 4 mM CaCl2) for 5 minutes on ice then centrifuged at 8000 x g for 5 minutes.  100 μl of lysate was mixed with TRIzol and saved as input.  To generate ribosome protected fragments the lysates were first mixed with 200 μl DEPC-H2O then incubated with 10 μg/ml micrococcal nuclease for 30 min at 37°C.  RNA was extracted with TRIzol LS.  T4 PNK was used to generate RNA fragments with a 5' phosphate and 3' hydroxyl.  The RNA was then run on a 15% 8M urea TBE gel, stained with SYBR Gold, and a gel fragment between 27-35 nucleotides corresponding to ribosome protected RNA was excised.  RNA was eluted overnight in 400 μl TE after crushing the gel fragment.  RNA was ethanol precipitated and resuspended in 7 μl water.  To generate libraries from RNA protected fragments 3 μl of the RNA was used with the NEBnext small RNA library prep kit (NEB) according to the manufacturer's instructions.  Due to the similarity in size between ligated and unligated adapters, prepared libraries (~155 nt) were gel purified. Ribosomal RNAs were removed from inputs using NEBNext rRNA Depletion Kit (NEB).  Input RNA libraries were generated using the NEBNext Ultra Directional RNA library prep kit for Illumina (NEB). Libraries were sequenced using an Illumina HiSeq 2500 platform with 50 nucleotide reads.