title: GSM3682395: Mt_TRAP_C4_R [MT-TRACON4]; Medicago truncatula; RNA-Seq
library_construction_protocol: TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (Townsley et al 2014) Random primer-primed RNA-seq library construction for nRNA (pre-rRNA and rRNA digested), polyadenylated total RNA and polyadenylated TRAP RNA was performed according the BrAD-seq method (Townsley et al 2014) Nuclei were purified from frozen and pulverized tissue as previously described for A. thaliana (Wang and Deal, 2015) with minor modifications including the use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract and extend centrifugation times. Tissue was resuspended in an ice-cold mortar containing 10 mL of freshly prepared nuclei purification buffer (NPB: 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, pH, 7.0) containing 200 uL Protease Inhibitor Cocktail (0.4X, Sigma, P9599) per 50 mL of buffer. The homogenized extracts were filtered through a 30 µM nylon mesh to remove cell debris and centrifuged at 1000 x g for 15 min at 4° C to pellet nuclei. Nuclei were resuspended in 1 mL of NPB and 25 µL of M-280 streptavidin-coated Dynabeads (Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated in a cold room at 4° C for 30 min. The nuclei/beads suspension was diluted to 14 mL with NPB supplemented with 0.1% (v/v) Triton X-100 (NPB-T), in a 15 mL Falcon tube, mixed thoroughly and placed in a 15 ml magnet (adapted NEB 50 mL tube magnet) to capture bead-bound nuclei for 1 min at 4° C. The supernatant was carefully removed using a plastic Pasteur pipette, taking care to remove bubbles to avoid disturbing the beads. Beads were resuspended in 14 ml of cold NPB-T, placed on a rotating mixer for 30 sec, and then placed back in the 15 ml magnet to capture the beads-nuclei at 4° C for 1 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using 20,000-50,000 nuclei as previously described (Maher et al 2017, Bajic et al 2018), with slight modifications. For rice, minor modifications in nuclei purification include: 1) the use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract, 2) extended centrifugation times (Reynoso et al 2018b, Reynoso et al 2018), and 3)using AMPureXP beads instead of columns to purify amplified libraries TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (5). Total RNA was extracted from frozen tissue using polysome extraction buffer (Mustroph et al 2009) followed by LBB polyA mRNA isolation using biotinylated oligodT and streptavidin magnetic beads (Townsley et al 2014) Random primer-primed RNA-seq library construction for nRNA (pre-rRNA and rRNA digested), polyadenylated total RNA and polyadenylated TRAP RNA was performed according the BrAD-seq method (Townsley et al 2014) in at least four biological replicates for each condition and species Ribo-seq libraries were generated as described by (Juntawong et al 2015) but with ribosome isolation by TRAP as described by (Bazin et al 2017) starting with pulverized frozen root tip tissue (~1000 1 cm root tip) thawed in 5 mL of Polysome Extraction Buffer and using ɑ-FLAG conjugated IgG Dynabeads for binding instead of anti-FLAG M2 magnetic beads. Manipulations were as previously described by (Bazin et al 2017) through to the generation of ribosome footprint fragments (RFs) and on-magnetic bead digestion of 1 mL of resuspended beads with 2000 units of RNase I (Ambion; ca. 15 U/μg RNA) by incubation for 180 min at 23-25 °C. RFs of  26-34 nt were gel purified, dephosphorylated using T4 polynucleotide kinase, ligated 500 ng preadenylylated miRNA cloning linker (IDT, miRNA cloning linker #1). The ligated-RFs were excised, recovered and resuspended in 10.0 μl of 10 mM Tris (pH 8). After this step, rRNA removal of RFs was done by use of Ribo-Zero rRNA Removal Kit (Plant; Illumina) probe solution
attributes.tag: GEO Accession
attributes.value: GSM3682395