Instrument: NextSeq 500
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For the RNase titration experiments, frozen tissues were thawed in ice-cold polysome lysis buffer [20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100µg/ml cycloheximide, 25U/ml Turbo DNaseI (Ambion, #AM2238), 1% NP-40 in nuclease-free water] and lysed by trituration through a 25-G need for 10 times. After 10-min incubation on ice, lysates were clarified by centrifugation at 2000g 4 °C for 10min. The supernatants were collected and clarified again by centrifugation at 20,000g 4 °C for 10min. The supernatants were collected and the amounts of nucleic acids were measured with Nanodrop (Thermo Fisher Scientific) and Qubit HS assays (Invitrogen). Lysate containing 2µg RNA in 0.3ml was digested with 5-fold serial diluted RNase A (Ambion, #AM2270) and RNAse T1 (Thermo Fisher Scientific, #EN0542) at 25°C for 30min, including RNase1 (4.8ng RNase A + 0.6U RNase T1/µg RNA), RNase2 (24ng RNase A + 3U RNase T1/µg RNA), RNase3 (120ng RNase A + 15U RNase T1/µg RNA), RNase4(600ng RNase A + 75U RNase T1/µg RNA), and RNase5 (3000ng RNase A + 375U RNase T1/µg RNA). The digestion was stopped by adding 50U SUPERase In RNase inhibitor (Ambion, #AM2694) and chilling on ice. Digested lysates were loaded on 10%-50% sucrose gradients prepared in 1Xpolysome buffer (20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100µg/ml cycloheximide in nuclease-free water). After the ultracentrifugation in a SW41Ti rotor (Beckman Coulter) at 35,000 rpm 4°C for 2.5 hours, gradients were fractionated at 1.5 ml/min and 12 sec collection interval through a fractionation system (Brandel) that continually monitored A260 values. Monosome fractions were identified, pooled, and extracted with TRIzol LS. For other samples, RNase A+T1 or RNase I were added as indicated based on the A260 units in the lysates. Libraries were prepared following the published protocols (Heyer et al., 2015; Ingolia et al., 2012). Briefly, rRNA was depleted from the purified monosomal RNA or cytoplasmic RNA samples with RiboZero (Illumina, #MRZG12324). For cyptoplamic RNA, RNA samples were fragmented in 1Xalkaline fragmentation solution at 95 °C for 15min. RNA samples were separated on a 15% TBU gel (National Diagnostics, #EC-833). Ribosome footprints were size-selected between 26-34nt, while cytoplamic RNAs were size-selected between 100-150nt.. RNA was eluted from the crushed gel pieces in RNA elution buffer (300mM NaOAc pH5.5, 1mM EDTA, 0.25% SDS) at RT overnight, filtered with Spin-X Centrifuge Tube Filters (Corning, #8162) and precipitated with equal volume of isopropanol. Recovered RNA was dephosphorylated with T4 Polynucleotide Kinase (NEB, #M0201S) and ligated with preadenylated adaptor in miRCat®-33 Conversion Oligos Pack (IDT) using T4RNL2Tr.K227Q ligase (NEB, #M0351L). Reverse transcription was performed with RT primers with 5nt-barcode and 5nt or 8nt unique molecular identifier (UMI) and SuperScript III (Invitrogen, #18080-044) in 1X first-strand buffer without MgCl2 (50 mM Tris-HCl, pH 8.3, 75 mM KCl). RT products were separated on a 10% TBU gel and the 130-140nt region was selected. cDNA was eluted in DNA elution buffer (10mM Tris pH 8.0, 300mM NaCl, 1mM EDTA) at RT overnight, filtered, and precipitated with isopropanol. Purified cDNA was circularized with CircLigase (Epicentre, #CL4115K). Optimal PCR cycle was determined empirically by test PCR reactions with titrated cycle numbers. Final PCR amplification was performed with KAPA Library Amplification Kit (Kapa Biosystems, #KK2611) and 180-190bp products were size-selected on an 8% TBE gel. DNA was eluted in DNA elution buffer and precipitated with isopropanol. The final library DNA was purified with AMPure XP beads (Beckman Coulter, #A63880). Ribosome profiling