GSM5310250: KO1 RPF; Homo sapiens; OTHER
Plates were washed with 10mL of ice-cold PBS with 100μg/mL cycloheximide twice, then cells were collected in ice-cold PBS and centrifuged at 500g for 5 minutes. Each pellet was resuspended in 3 pellet volumes of lysis buffer (20mM HEPES pH 7.6, 100mM KCl, 5mM MgCl2, 1% Triton x-100, 100μg/mL cycloheximide, 1% v/v SUPERaseIN inhibitor in nuclease-free water) and cells were lysed with rotation at 4ºC for 20 minutes before being centrifuged at 16,000 g for 15 minutes. To the clarified lysate, 4μL of Turbo DNase (Thermo Fisher) was added and samples were incubated at room temperature 15 minutes then centrifuged again to clear the lysate. About 20% of the sample was saved as input with 3 volumes of Trizol added, while the other 80% was treated with 3μL MNase (NEB) in 1x MNase buffer for 15 minutes at room temperature to digest RNA not covered by ribosomes. Additional SUPERaseIN (Invitrogen) was added to a final concentration of 0.5mM to quench the reaction. Then, samples were loaded on 5-50% w/v sucrose gradients and fractionated by Gradient Station (BioComp) equipped with an ECONO UV monitor (BioRad) and fraction collector (Gilson). Fractions containing the digested monosomes were collected and pooled, and three volumes of Trizol (Invitrogen) was added to each sample. RNA purification was performed for both input and ribosome-protected fragment (RPF) samples by chloroform extraction followed by isopropanol precipitation, and one round of rRNA depletion was performed (see “RNA purification” section of Methods). Input RNA was fragmented by combining 20μL RNA with 7.5 uL PNK buffer A (Thermo Fisher) and incubating at 94ºC for 25 minutes, then end repaired by diluting in 37.5μL nuclease-free water, adding 10μL T4 PNK (Thermo Fisher) and incubating at 37ºC for 30 minutes. Then, 12μL 10mM ATP (EMD Millipore), 2μL 10x PNK buffer A, and 6μL T4 PNK were added to incubate at 37ºC for 30 minutes. These samples were purified using the RNA clean and concentrator-5 kit (Zymo Research). Purified RPF RNA was run in 1x Novex TBU sample buffer (Thermo Fisher) on a 10% TBE-urea gel (Thermo Fisher) in 1x RNase-free TBE buffer (Thermo Fisher) with an IDT 10/60 loading control (IDT) for 1 hour at 180V. The gel was stained 15 minutes in 1x SYBR gold in TBE with gentle shaking and visualized using a Biorad Gel Doc EZ Imager. Fragments between 26-34 nucleotides were excised and purified by Small-RNA PAGE recovery kit (Zymo Research) per manufacturer's instructions. RPFs were end repaired similarly to inputs with T4 PNK. Input and RPF RNA samples were used to prepare libraries for sequencing using the NEBNext Small RNA Library Prep Kit (NEB) per manufacturer's instructions, and were sequenced by Illumina NovaSeq6000 with 100bp single-end reads.