Instrument: NextSeq 500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Frozen cell pellets were thawed in ice-cold polysome lysis buffer [20mM Tris-HCl pH7.4, 5mM MgCl2, 100mM KCl, 1mM DTT, 100ug/ml cycloheximide, 25U/ml Turbo DNaseI (Ambion, #AM2238), 1% TritonX-100 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 14,000g 4 °C for 10min. The supernatants were collected and the amounts of nucleic acids (A260 units) were measured with Nanodrop (Thermo Fisher Scientific). For each sample, cytoplasmic RNA for RNA-seq was purified from one fourth of the lysate with TRIzol LS reagent (Invitrogen, #10296028). The other three fourths of the lysate was digested with 100ng/A260 RNase A (Ambion, #AM2270) and 60U/A260 RNAse T1 (Thermo Fisher Scientific, #EN0542) at 25°C for 30min. 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, 100ug/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. Ovation RNA‑Seq System V2 (NuGEN) For the cytoplasmic RNA-seq, 500ng total RNA per sample was used for the library preparation with the Ovation RNA‑Seq System V2 (NuGEN, #0304) following manufacturer's instructions. HL-dsDNase (ArcticZymes, #70800-201) method was used to eliminate DNA contamination and cDNA was fragmented with M220 Covaris Focused-ultrasonicator for a target median fragment size of 300bp (peak power: 50.0, duty factor 20.0, cycles/burst: 200, treatment time: 75sec). qPCR test was performed with EvaGreen Dye (Biotium, #31000-T) to determine the optimal PCR cycles. For the ribosome profiling samples, libraries were prepared following the published protocols (Heyer et al., 2015; Ingolia et al., 2012). Briefly, rRNA was depleted from the purified monosomal RNA samples with RiboZero (Illumina, #MRZG12324). Remaining RNA samples were separated on a 15% TBU gel (National Diagnostics, #EC-833) and the ribosome footprints were size-selected based on the 26 and 34nt markers. 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). cDNA derived from remaining rRNA was hybridized to biotin-labelled antisense probes (IDT) and further depleted with Dynabeads MyOne Streptavidin C1 (Inivtrogen, #65001). 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)