title: GSM8807005: RPF_rep1_EME; Homo sapiens; OTHER
library_construction_protocol: At the time of collection, 10-35% sucrose gradient was prepared in ultra-centrifuge tolerant tube (Seton) by combining 10% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 10% sucrose, 0.1% protease inhibitor, 0.1% phosphatase inhibitor) with 35% sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 35% sucrose, 0.1% protease inhibitor, 0.1% phosphatase inhibitor)using Gradient Station (Biocomp). The sucrose gradient for all samples was stored in cold room (4 ºC) before use. Cells in 4× 15-cm plates were then treated with 1 µg/ml emetine (positive control) for 15 min followed by immediate wash with ice-cold 1× PBS buffer. Cells were immediately collected with 10 ml ice-cold 1× PBS and pelleted by centrifugation at 500× g for 5 min. Cells from 2× 15-cm plates were combined as one sample. 4× volumes of lysis buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 1% Triton X-100, 1% protease inhibitor, 1% phosphatase inhibitor) were added to the combined cell pellet and the cells were lysed on ice for 20 min with periodic perturbation (or rotating at 4 ºC). Supernatant lysate was collected after centrifugation at 16,000× g for 15 min. 4 µl Turbo DNase (ThermoFisher) was added to the lysate and incubated at room temperature for 15 min. Supernatant lysate was collected after centrifugation at 16,000× g for 15 min after DNase treatment to remove any debris. Total RNA concentration in the lysate was measured using the Qubit RNA HS Assay Kit (ThermoFisher, Q32852) after 20× dilution according to the manufacturer's manual. Lysate containing 90 µg of total RNA from each sample was used and adjusted to 500 µl using the lysis buffer. 5 µl 100 mM CaCl2 was added to each sample to 1 mM final. 1 µl 100 U/µl Micrococcal Nuclease (Thermo Fisher, 88216) was added to each sample and mixed. RNA digestion was performed for 40 min at 25 °C before the reaction was terminated by adding 10 µl SUPERaseIn RNase inhibitor (ThermoFisher, AM2696). For Ribo-seq, lysate after RNase digestion (~516 µl) together with 8 ml sucrose cushion buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 0.1 % SUPERaseIn RNase inhibitor (ThermoFisher), 0.1% protease inhibitor, 0.1% phosphatase inhibitor) was loaded gently to the bottom of the 70.1 Ti ultracentrifuge tubes (Beckman Coulter, 355603). 1.5 ml 37.5% (1M) sucrose buffer (20 mM HEPES (pH 7.6), 100 mM KCl, 5 mM MgCl2, 37.5% sucrose, 0.1 % SUPERaseIn RNase inhibitor (ThermoFisher), 0.1% protease inhibitor, 0.1% phosphatase inhibitor) was loaded gently to the bottom of the tube using long blunt end needle. All the ultracentrifuge tubes were weighed and balanced. The tubes were then centrifuged at 65,000 rpm and 4 ºC under vacuum for 2.5 hours using ultracentrifuge (Beckman Coulter Optima L-100XP, 70.1 Ti rotor). After ultracentrifugation, the supernatant was removed, and the ribosome pellet was washed with 2 ml sucrose cushion buffer three times. 1 ml TRIzol reagent was directly added to the ribosome pellet and incubated at room temperature for 10 mins or until the ribosome pellet was completely dissolved. The TRIzol ribosome mixture was then transferred to microcentrifuge tubes and total RNA was extracted according to manufacturer's manual. 5 µg RNA from each sample was loaded on 10% TBE-Urea gel for ribosome protected fragments extraction. For both monosome and disome protected fragments, gel region containing RNA from 15 to 40 nt and 50 to 80 nt was excised from the gel and transferred to microcentrifuge tubes. The gel slices were crushed into tiny pieces using pipet tips or disposable homogenizer pestle. 400 µl of RNA gel extraction buffer (300 mM NaOAc, pH 5.2, 1 mM EDTA, 0.25% SDS) was added to the gel pieces and flash-freeze by liquid nitrogen. The samples were then mixed end-to-end on a rotary mixer at room temperature for 12-20 hours. The RNA gel extraction buffer and gel pieces mixture was transferred to a 0.22-µm filter inserted into a 1.5-ml microcentrifuge tube (Corning Spin-X Centrifuge Tube Filters, 8160) using wide-bore tips. The samples were centrifuged at 10,000× g at 4 ºC for 1 min. The cleared elute was transferred to new microcentrifuge tubes. 3 µl of GlycoBlue and 500 µl isopropanol were added to each sample and thoroughly mixed followed by incubation in -20 °C freezer overnight. Samples were then centrifuged at 17,000 × g at 4 °C for 30 min to pellet the RNA. The RNA pellets were washed with 1 ml of 75% ethanol followed by centrifuge at 4 °C for 3 min. Supernatant was completely removed and RNA pellets were air-dried for 10 min at room temperature. RNA was resuspended in 41 µl H2O and the concentrations were measured using the Qubit RNA HS Assay Kit (ThermoFisher). 5 µl 10× T4 PNK buffer A and 5 µl T4 PNK (ThermoFisher, EK0032) were added to the RPF RNA from above (~40 µl) and mixed. The samples were incubated at 37 °C for 20 min. 6 µl 10 mM ATP, 1 µl 10× T4 PNK buffer A, and 3 µl T4 PNK were then added to each sample (~60 µl total) and mixed followed by incubation at 37 °C for 20 min. 120 µl RNA binding buffer and 270 µl 100% ethanol were added to each sample followed by RNA purification using RNA Clean & Concentrator-5 (Zymo, R1016). The purified RNA samples were then used for sequencing library construction using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S) according to manufacturer's manual. rRNA blocker oligos (Nanodigmbio 1002901) were added at the reverse transcription primer hybridization step during library construction to reduce rRNA reads. Sequencing libraries were sequenced on Illumina NextSeq system using single-end kit (Illumina, 20100995). mRNA inputlibraries for ribo-seq was constructed using MSR-seq (PMID: 35513407). NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S) and MSR-seq (PMID: 35513407)
identifiers.namespace: GEO
identifiers.id: GSM8807005_r1