GSM8322255: DDX1 ON NCC rep3; Homo sapiens; OTHER
cNCCs derived from three separate differentiations were rapidly washed twice with ice-cold PBS supplemented with 100 μg/mL cycloheximide and lysed on-plate in 500 μL ice-cold Polysome Lysis Buffer (20mM HEPEs pH 7.3, 100mM KCl, 5mM MgCl, 1% Tritonx-100, 2 μM DTT, 100 μg/mL cycloheximide). Lysate was passed through a 26 gauge needle 10 times and clarified by centrifugation for 5 minutes at 1,300 x g, 4°C, and the supernatant was further clarified by centrifugation for 5 minutes at 20,000 x g, 4°C. Samples were normalized to the same concentration, as measured by NanoDrop Spectrophotometer, and treated with 350 U Ambion RNAseI (Invitrogen) per O.D. of absorbance at 260 nM per mL for 16 hours at 4°C. Samples were loaded on cold 10-50% sucrose gradients generated by layering 6 mL of 10% sucrose gradient buffer (20 mM HEPES pH 7.3, 100 mM KCl, 5 mM MgCl, 1 mM DTT, 100 μg/mL cycloheximide, 10% sucrose w/v) on 6 mL of 50% sucrose gradient buffer (20 mM HEPES pH 7.3, 100 mM KCl, 5 mM MgCl, 1 mM DTT, 100 μ/mL cycloheximide, 50% sucrose w/v) and mixed using a BioComp Gradient Station (BioComp, New Brunswick, Canada). Sample-loaded gradients were spun at 210,000 x g for 2.5 hours at 4°C using a SW-41 swinging-bucket rotor (Beckman Coulter) and Optima XPN Ultracentrifuge (Beckman Coulter). These gradients were fractionated on a BioComp Gradient Station and the monosome peaks were collected. RNA was extracted from the collected monosome peaks using Trizol LS (Invitrogen) and purified using a Direct-zol RNA Miniprep kit (Zymo Research). Fragments between 17-34 nt long were isolated and libraries were prepared from them as described in McGlincy and Ingolia (2017), except with a depletion of rRNA immediately before reverse transcription using a mixture of rRNA-complementary biotinylated oligos (http://bartellab.wi.mit.edu/protocols.html) Libraries were sequenced with a 75 nt single-end protocol on a 150 nt high output flowcell of an Aviti sequencer (Element Biosciences) after Adept library conversion (Element Biosciences).