title: GSM5554172: Col-0 seedling RNA-seq sample 1; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Plant lysates were prepared as previously described. Briefly, per 0.1 g of grounded tissue power was resuspend in 400 µL of lysis buffer (100 mM Tris-HCl [pH 8], 40 mM KCl, 20 mM MgCl2, 2% [v/v] polyoxyethylene [10] tridecyl ether [Sigma, P2393], 1% [w/v] sodium deoxycholate [Sigma, D6750], 1 mM dithiothreitol, 100 µg/mL cycloheximide [Sigma, C4859], 100 µg/mL chloramphenicol [Sigma R4408], and 10 units/mL DNase I [Epicenter, D9905K]). The lysates were spined at 3,000 g for 3 min and the supernantant was transferred to new tube and subsequently centrifuged at 20,000 g for 10 min. The supernantant was transferred to new tube and the RNA concentration was determined using Qubit RNA HS assay (Thermo Fisher Scientific; Q32852). Aliquots of 100 µL and 200 µL of the lysates were made and flash frozen in liquid nitrogen and stored in -80°C freezer until further process Ribo-seq library construction: Ribosome footprints were obtained using 200 µL of the lysates above and the sequencing libraries were constructed following our previous method with the following modifications: briefly, after RNase I digestion (50 unit nuclease per 40 µg of RNA, the nuclease was provided in TruSeq Mammalian Ribo Profile Kit, illumina, RPHMR12126) and size exclusion column step, RNA > 17 nt was isolated by RNA Clean & Concentrator-25 kit (Zymo Research, R1017) and separated in 15% Urea-TBE gels (Invitrogen, EC68852BOX). Gel slices between 28-30 nt were isolated and the RNAs were purified as previously described. Next, rRNA depletion was performed using RiboZero Plant Leaf kit (Illumina, MRZPL1224) with one quarter of recommended reaction volume. Ribo-seq libraries were then constructed using TruSeq Mammalian Ribo Profile Kit (illumina, RPHMR12126) as previously described with 9 cycles of PCR amplification. Equal molarity of libraries were pooled and sequenced on Hi-Seq 4000 using single end 50 bp sequencing. RNA-seq library construction: Total RNA greater than 200 nt was purified using 100 µL of the lysates above as previously described with RNA Clean & Concentrator-25 kit (Zymo Research, R1017). RNA integrity was evaluated using Bioanalyzer (Agilent) RNA pico chip and RNA integrity number (RIN) ranging from 7.2 to 7.7 were observed among the samples. A 4 µg of RNA per sample was subjected to rRNA depletion using RiboZero Plant Leaf kit (Illumina, MRZPL1224) following the manufacturer's recommendation. Then, 100 ng of rRNA-depleted RNA was fragmented to around 200-nt long based on the RIN reported by Bioanalyzer, and strand-specific sequencing libraries were made using NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7760S) with 8 cycles of amplification. Equal molarity of libraries were pooled and sequenced on Hi-Seq 4000 using pair-end 100 bp sequencing.