title: GSM2193124: GRO-seq rep1; Arabidopsis thaliana; OTHER
library_construction_protocol: For RNA-seq, total RNA was extracted from seedlings frozen in liquid nitrogen using RNeasy Plant Mini Kit (Qiagen). 10µg of total RNA was used for extraction of mRNA with the Poly(A)Purist MAG Kit (Ambion) according to manufacturer’s instructions. For GRO-seq/5'GRO-seq, approximately 20 grams of 6-day-old seedlings were homogenized with an OMNI International General Laboratory Homogenizer in ice cold grinding buffer (300 mM sucrose, 20 mM Tris, pH 8.0, 5 mM MgCl2, 5 mM KCl, 0.2% Triton X-100, 5 mM β-mercaptoethanol, 35% glycerol) at 4°C on low speed. Samples were filtered twice through a 250 micron nylon mesh and then miracloth before being split into 50ml conical tubes and spun for 10 minutes at 5000g. Supernatant was discarded, and the pellets were resuspended in grinding buffer using a Kimble loose dounce homogenizer (Fisher Scientific). Wash step was repeated twice. Nuclei were resuspended in freezing buffer (50 mM Tris, pH 8.0, 5 mM MgCl2, 20% glycerol, 5 mM β-mercaptoethanol) and frozen in liquid N2. Approximately 5x106 nuclei were used for run-ons at room temperature for 5’ and CTP concentration was limited to 20 nM. After run-on, samples were treated with RQ1 DNaseI (Promega) for 20 minutes at 37°C followed by Trizol LS (Fisher Scientific) extraction as described by the manufacturer. Samples were then treated with Terminator 5’-Phosphate-Dependent Exonuclease (Epicentre) for 1 hour at 30°C. RNA fragmentation was carried out with Fragmentation Reagents kit (Ambion) for 11’ at 70°C. Treatments were used to deplete ribosomal and chloroplast RNA. Nascent RNA was enriched by double pull down using BrdU antibody beads (Santa Cruz Biotechnology) for 80’ at 4C, washed three times with GRO-Binding buffer (0.25x SSPE, 0.05% Tween, 37.5 mM NaCl , 1 mM EDTA) then extracted with Trizol LS (Fisher Scientific). Isolated mRNA was fragmented with Fragmentation Reagents (Ambion) for 14’. Fragmented RNA was incubated with T4 polynucleotide kinase (New England Biolabs) in low pH buffer (0.1 M MES, 10 mM MgCl2, 10 mM mercaptoethanol, 300 mM NaCl - pH 5.6) for 3’ repair, then T4PNK buffer for 5’ phosphorylation. For 5'GRO-seq, de-capping using TAP was replaced with RppH (New England Biolabs) and NEB Next Small RNA Library Prep Set (New England Biolabs) was used for library preparation. De-capping was performed in 3’ Ligation buffer at 37°C in the absence of 3’ adapter. 3’Adapter ligation was performed at 20°C instead of 25°C so RppH is inactive. The Small RNA Library Prep Set was (New England Biolabs) was used for final library preparation of short RNA fragments from each method. Strand specific polyA RNA-seq, GRO-seq, and 5'GRO-seq
attributes.tag: GEO Accession
attributes.value: GSM2193124