title: GSM3560215: Root Ribo-seq sample 2; Solanum lycopersicum; OTHER
library_construction_protocol: The frozen tissues were pulverized with liquid nitrogen using a pestle and a mortar. About 0.4 g tissue powder was resuspended in 1.2 ml lysis buffer described in Hsu et al. 2016 (100 mM Tris·HCl (pH 8), 40 mM KCl, 20 mM MgCl2, 2% (vol/vol) polyoxyethylene (10) tridecyl ether (Sigma P2393), 1% sodium deoxycholate (Sigma D6750), 1 mM DTT, 100 μg/mL cycloheximide, and 10 unit/mL DNase I (Epicenter D9905K)). After incubation on ice with gentle shaking for 10 minutes, the lysate was spun at 4°C at 20,000 g for 10 minutes. The supernatant was transferred to new tube, and 100 µL and 200 µL aliquots were created. The aliquoted lysates were flash frozen with liquid nitrogen and stored at -80°C until ready to be processed. For RNA-seq samples, 10 µL 10% SDS was added to the above 100 µL lysate. RNA greater than 200 nt was extracted using Zymo RNA clean and concentrator kit (Zymo Research R1017). The obtained RNA was checked by a Bioanalyzer (Agilent) RNA pico chip to access the RNA integrity, and a RIN value ranging from 9.2 to 9.4 was obtained for each replicate. Ribosomal RNAs (rRNAs) were depleted using RiboZero Plant Leaf kit (Epicenter/illumina MRZPL1224). Next, 100 ng of the rRNA-depleted RNA was used as the starting materials and fragmented to ~200 nt size based on the RIN reported by Bioanalyer following the NEBNext Ultra Directional RNA Library Prep Kit (NEB E7420S) to create strand-specific libraries. The libraries were barcoded and enriched using 11 cycles of PCR amplification. Equal molarity of the libraries was pooled and sequenced using Hi-Seq 4000 PE-100. The Ribo-seq samples were prepared according to Hsu et al. 2016 with minor modifications. Briefly, the RNA concentration of each lysate was first determined by Qubit RNA HS assay using a 10-fold dilution. Next, the 200 µL lysate above was treated with 100 units of Nuclease (provided by the TruSeq Mammalian Ribo Profile Kit, illumina RPHMR12126) per 40 µg of RNA with gently shaking at room temperature for one hour. The nuclease reaction was stopped by immediately transferred to ice and adding 15 µL of SUPERase-IN (Invitrogen AM2696). The ribosomes were isolated using illustra MicroSpin S-400 HR Columns (GE Healthcare 27514001). The RNA greater than 17 nt was purified first, and then RNA smaller than 200 nt was enriched using the RNA Clean & Concentrator Kit (Zymo Research R1017). Next, the rRNAs were depleted using the RiboZero Plant Leaf kit (Epicenter/illumina MRZPL1224). The rRNA-depleted RNA was then separated by 15% (wt/vol) TBE-urea PAGE (Invitrogen EC68852BOX), and gel slices ranging from 28 to 30 nt were excised. Ribosome footprints were recovered from the excised gel slices following the overnight elution method and the sequencing libraries were constructed according to the TruSeq Mammalian Ribo Profile Kit manual. The final libraries were amplified by 9 cycles of PCR. Equal molarity of the libraries was pooled and sequenced using Hi-Seq 4000 SE-50.
attributes.tag: GEO Accession
attributes.value: GSM3560215