title: GSM4212888: RNA_Ctrl_rep2; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Whole shoot tissues of 14-day-old seedlings (approximately 300 individuals per replicate) were harvested and frozen immediately with liquid nitrogen. For Ribo-seq, Tissue was frozen in liquid nitrogen, powdered by using a chilled mortar and pestle, and homogenized in ice-cold polysome extraction buffer [100 mM Tris-HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 μg/mL cycloheximide, 2% polyoxyethylene (10) tridecyl ether, 1% deoxycholic acid, 10 U/mL DNase I (Qiagen, Hilden, Germany)]. After incubation on ice for 10 min, tissues were homogenized on ice by using a tight-fitting Dounce homogenizer. The homogenates were centrifuged for 30 min at 16,000g and 4 °C in a JA25.50 rotor (Beckman Coulter, California, United States) to pellet the insoluble cell debris. The supernatant was layered on top of a 1.75-M sucrose cushion [100 mM Tris-HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 μg/mL cycloheximide, 1.75 M sucrose] and centrifuged for 18 h at 170,000g and 4 °C in a TY70 Ti rotor (Beckman) to obtain a polysome pellet. The pellet was resuspended in RNase I digestion buffer [100 mM Tris-HCl (pH 8.0), 40 mM KCl, 20 mM MgCl2, 1 mM dithiothreitol, 100 μg/mL cycloheximide]. The OD260 was determined by using a Nanodrop1000 spectrophotometer (Thermo Fisher Scientific, Massachusetts, United States). Then, 2000 OD units of polysome was digested with 2000 units of RNase I (Thermo Fisher Scientific) in a 185-L reaction mixture for 2 h at 25 °C with gentle rotation. Digestion was stopped by adding 15 L of SUPERase-In RNase inhibitor (Thermo Fisher Scientific). Monosome isolation in illustra MicroSpin S-400 HR columns (GE Healthcare, Illinois, United States), RNA extraction and concentration by using an RNA Clean & Concentrator kit (Zymo Research, California, United States), and rRNA depletion with a Ribo-Zero Plant Leaf kit (Illumina, California, United States) were performed as described by Hsu PY, et al. (Proceedings of the National Academy of Sciences, 2016, 113.45, E7126-E7135.). For RNA-seq, total RNA was extracted by using an RNeasy Plant Mini Kit with DNase I treatment on a column in accordance with the manufacturer's protocol (Qiagen). For Ribo-seq, purified RNA was separated by 12% 7M urea PAGE in TBE buffer, and gel slices from 28 to 30 nt long were excised. Ribosome footprint extraction from the gel slice and subsequent Ribo-seq library construction and amplification by PCR were performed as described in the manufacturer's protocol [TruSeqRibo Profile (Mammalian) kit, Illumina]. Libraries #1 and #2 were amplified by 11 and 13 cycles, respectively, of PCR.  For RNA-seq, ibrary preparation and sequencing were performed by a commercial service (BGI, Shenzhen, China).
attributes.tag: GEO Accession
attributes.value: GSM4212888