GSM2730478: H10m_rice_42oC_10min_riboseq_rep2.fastq.gz; Oryza sativa; OTHER
The RPFs (ribosome protected fragments) isolation and library construction were done as described in the literature45 with some major changes. Rice shoots were grounded into powder with liquid nitrogen. Two mL of tissue powder was used for each sample and then dissolved and homogenized in 10 ml polysome extraction buffer. The buffer contains 200 mM Tris-Cl (pH 8.0), 100 mM KCl, 25 mM MgCl2, 5 mM DTT, 1 mM PMSF, 100 μg/mL cycloheximide, 1% Brj-35, 1% TritonX-100, 1% Igepal CA630, 1% Tween-20, 1% polyoxyethylene 10 tridecyl ether. After centrifugation at 16 000 g for 10 min at 4 °C, the supernatant was collected. The supernatant was then layered on top of an 8 mL sucrose cushion (1.75 M sucrose in 200 mM Tris (pH 8.0), 100 mM KCl, 25 mM MgCl2, 5 mM DTT, 100 μg/mL cycloheximide), and centrifuged at 170 000 g at 4 °C for 3 h. The pellet was resuspended in 400 μL RNase I digestion buffer (50 mM Tris-Cl (pH 8.0), 100 mM KCl, 20 mM MgCl2, 1 mM DTT and 100 μg/mL cycloheximide). After adding 20 μL RNase I (Cat# AM2294, Thermo Fisher), RNase digestion was performed at room temperature with rotation for 2 h. TRIzol reagent (Cat# 15596026, Thermo Fisher) was used to extract the RPFs followed by fragment size selection using NucleoSpin miRNA kit (Cat# 740971, Macherey Nagel) to collect the fragments smaller than 200 nt. 10% of Urea-PAGE gel was then applied to size select 28-32 nt fragments. After dephosphorylation using PNK (Cat# M0201S, NEB), the RPFs were ligated to AIR adenylated RNA linker (Cat# 510201, BIOO Scientific). The ligation products were then subjected to reverse transcription using SSIII (Cat# 18080093, Thermo Fisher) and circularization using Circligase II (Cat# CL9021K, Illumina). Sequence libraries were ultimately obtained through PCR amplification by Q5 polymerase (Cat# M0491S, NEB). Ribosome profiling libraries were sequenced to generate single-end 100 nt reads done by the Genomics Core Facility at Penn State University. Ribo-seq
GEO Accession
GSM2730478