alias: GSM2889182
title: GSM2889182: GROseq_ago4_D762A_rep2; Arabidopsis thaliana; OTHER
library_construction_protocol: Approximately 10 grams of inflorescence and meristem tissue was collected from plants and immediate placed 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). Nuclei were isolated as described previously16. Briefly, samples were ground with an OMNI International General Laboratory Homogenizer at 4°C until well homogenized, filtered through a 250µm nylon mesh, 100µm nylon mesh, miracloth, and finally a 40µm cell strainer before being split into 50ml conical tubes.  Samples were spun for 10 minutes at 5250g, the supernatant was discarded, and the pellets were pooled and resuspended in 25ml of grinding buffer using a dounce homogenizer.  The wash step was repeated at least once more and nuclei were resuspended in 1ml of freezing buffer (50 mM Tris, pH 8.0, 5 mM MgCl2, 20% glycerol, 5 mM β-mercaptoethanol).Approximately 5x106 nuclei in 200 µl of freezing buffer were run-on in 3x GRO-reaction buffer 16 with. Reactions were stopped after 5 minutes to minimize run on length (~5-15 nts) while still incorporating brUTP by addition of 750 µl Trizol LS and RNA purified according to the manual. Without fragmentation or Terminator treatment, nascent RNA was enriched twice for BrU by αBrdU (Santa Cruz Biotechnology sc-32323AC) and immunoprecipitated as described in Hetzel et al. 2016. Sequencing libraries were prepared from precipitated RNA using TruSeq Small RNA Library Prep kit following manufacturer instructions (Illumina). For GROseq in Col-0, nrpd1, nrpe1 and nrpd1/e1 double mutants, 14 cycles of PCR were used to amplify the libraries and the products ranging from 100 to 500bp were size selected by agarose gel. The libraries were sequenced on Illumina HiSeq 2000 platform by paired-end 100bp sequencing. For GROseq in spt5l, 14 cycles of PCR were used to amplify the libraries and the products were size selected by double SPRI bead purification (ratio of Ampure beads to library: 0.5:1 to 1.1:1).
identifiers.namespace: GEO
identifiers.id: GSM2889182