SRX5554177 GSM3682208 SRP189094 SRS4518072 GSM3682208 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682208 GSM3682208 GEO Accession GSM3682208 SRX5554178 GSM3682209 SRP189094 SRS4518074 GSM3682209 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682209 GSM3682209 GEO Accession GSM3682209 SRX5554179 GSM3682210 SRP189094 SRS4518073 GSM3682210 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682210 GSM3682210 GEO Accession GSM3682210 SRX5554180 GSM3682211 SRP189094 SRS4518075 GSM3682211 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682211 GSM3682211 GEO Accession GSM3682211 SRX5554181 GSM3682212 SRP189094 SRS4518076 GSM3682212 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682212 GSM3682212 GEO Accession GSM3682212 SRX5554182 GSM3682213 SRP189094 SRS4518077 GSM3682213 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682213 GSM3682213 GEO Accession GSM3682213 SRX5554183 GSM3682214 SRP189094 SRS4518078 GSM3682214 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682214 GSM3682214 GEO Accession GSM3682214 SRX5554184 GSM3682215 SRP189094 SRS4518079 GSM3682215 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682215 GSM3682215 GEO Accession GSM3682215 SRX5554185 GSM3682216 SRP189094 SRS4518081 GSM3682216 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682216 GSM3682216 GEO Accession GSM3682216 SRX5554186 GSM3682217 SRP189094 SRS4518080 GSM3682217 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682217 GSM3682217 GEO Accession GSM3682217 SRX5554187 GSM3682218 SRP189094 SRS4518082 GSM3682218 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682218 GSM3682218 GEO Accession GSM3682218 SRX5554188 GSM3682219 SRP189094 SRS4518083 GSM3682219 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682219 GSM3682219 GEO Accession GSM3682219 SRX5554189 GSM3682220 SRP189094 SRS4518084 GSM3682220 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682220 GSM3682220 GEO Accession GSM3682220 SRX5554190 GSM3682221 SRP189094 SRS4518085 GSM3682221 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682221 GSM3682221 GEO Accession GSM3682221 SRX5554191 GSM3682222 SRP189094 SRS4518086 GSM3682222 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682222 GSM3682222 GEO Accession GSM3682222 SRX5554192 GSM3682223 SRP189094 SRS4518087 GSM3682223 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682223 GSM3682223 GEO Accession GSM3682223 SRX5554193 GSM3682224 SRP189094 SRS4518088 GSM3682224 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682224 GSM3682224 GEO Accession GSM3682224 SRX5554194 GSM3682225 SRP189094 SRS4518090 GSM3682225 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682225 GSM3682225 GEO Accession GSM3682225 SRX5554195 GSM3682226 SRP189094 SRS4518089 GSM3682226 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682226 GSM3682226 GEO Accession GSM3682226 SRX5554196 GSM3682227 SRP189094 SRS4518091 GSM3682227 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682227 GSM3682227 GEO Accession GSM3682227 SRX5554197 GSM3682228 SRP189094 SRS4518092 GSM3682228 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682228 GSM3682228 GEO Accession GSM3682228 SRX5554198 GSM3682229 SRP189094 SRS4518093 GSM3682229 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682229 GSM3682229 GEO Accession GSM3682229 SRX5554199 GSM3682230 SRP189094 SRS4518094 GSM3682230 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682230 GSM3682230 GEO Accession GSM3682230 SRX5554200 GSM3682231 SRP189094 SRS4518095 GSM3682231 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682231 GSM3682231 GEO Accession GSM3682231 SRX5554201 GSM3682232 SRP189094 SRS4518096 GSM3682232 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682232 GSM3682232 GEO Accession GSM3682232 SRX5554202 GSM3682233 SRP189094 SRS4518097 GSM3682233 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682233 GSM3682233 GEO Accession GSM3682233 SRX5554203 GSM3682234 SRP189094 SRS4518099 GSM3682234 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682234 GSM3682234 GEO Accession GSM3682234 SRX5554204 GSM3682235 SRP189094 SRS4518098 GSM3682235 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682235 GSM3682235 GEO Accession GSM3682235 SRX5554205 GSM3682236 SRP189094 SRS4518100 GSM3682236 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682236 GSM3682236 GEO Accession GSM3682236 SRX5554206 GSM3682237 SRP189094 SRS4518101 GSM3682237 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682237 GSM3682237 GEO Accession GSM3682237 SRX5554207 GSM3682238 SRP189094 SRS4518102 GSM3682238 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682238 GSM3682238 GEO Accession GSM3682238 SRX5554208 GSM3682239 SRP189094 SRS4518103 GSM3682239 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682239 GSM3682239 GEO Accession GSM3682239 SRX5554209 GSM3682240 SRP189094 SRS4518104 GSM3682240 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682240 GSM3682240 GEO Accession GSM3682240 SRX5554210 GSM3682241 SRP189094 SRS4518105 GSM3682241 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682241 GSM3682241 GEO Accession GSM3682241 SRX5554211 GSM3682242 SRP189094 SRS4518106 GSM3682242 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682242 GSM3682242 GEO Accession GSM3682242 SRX5554212 GSM3682243 SRP189094 SRS4518107 GSM3682243 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682243 GSM3682243 GEO Accession GSM3682243 SRX5554213 GSM3682244 SRP189094 SRS4518108 GSM3682244 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682244 GSM3682244 GEO Accession GSM3682244 SRX5554214 GSM3682245 SRP189094 SRS4518110 GSM3682245 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682245 GSM3682245 GEO Accession GSM3682245 SRX5554215 GSM3682246 SRP189094 SRS4518109 GSM3682246 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR6 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682246 GSM3682246 GEO Accession GSM3682246 SRX5554216 GSM3682247 SRP189094 SRS4518112 GSM3682247 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR4 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682247 GSM3682247 GEO Accession GSM3682247 SRX5554217 GSM3682248 SRP189094 SRS4518111 GSM3682248 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR4 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682248 GSM3682248 GEO Accession GSM3682248 SRX5554218 GSM3682249 SRP189094 SRS4518113 GSM3682249 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR4 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682249 GSM3682249 GEO Accession GSM3682249 SRX5554219 GSM3682250 SRP189094 SRS4518114 GSM3682250 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR4 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682250 GSM3682250 GEO Accession GSM3682250 SRX5554220 GSM3682251 SRP189094 SRS4518115 GSM3682251 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682251 GSM3682251 GEO Accession GSM3682251 SRX5554221 GSM3682252 SRP189094 SRS4518116 GSM3682252 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682252 GSM3682252 GEO Accession GSM3682252 SRX5554222 GSM3682253 SRP189094 SRS4518117 GSM3682253 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682253 GSM3682253 GEO Accession GSM3682253 SRX5554223 GSM3682254 SRP189094 SRS4518118 GSM3682254 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682254 GSM3682254 GEO Accession GSM3682254 SRX5554224 GSM3682255 SRP189094 SRS4518119 GSM3682255 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682255 GSM3682255 GEO Accession GSM3682255 SRX5554225 GSM3682256 SRP189094 SRS4518120 GSM3682256 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682256 GSM3682256 GEO Accession GSM3682256 SRX5554226 GSM3682257 SRP189094 SRS4518121 GSM3682257 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:HF-FLAG-OsRPL8 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009). Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682257 GSM3682257 GEO Accession GSM3682257 SRX5554227 GSM3682258 SRP189094 SRS4518122 GSM3682258 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682258 GSM3682258 GEO Accession GSM3682258 SRX5554228 GSM3682259 SRP189094 SRS4518123 GSM3682259 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682259 GSM3682259 GEO Accession GSM3682259 SRX5554229 GSM3682260 SRP189094 SRS4518124 GSM3682260 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682260 GSM3682260 GEO Accession GSM3682260 SRX5554230 GSM3682261 SRP189094 SRS4518125 GSM3682261 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682261 GSM3682261 GEO Accession GSM3682261 SRX5554231 GSM3682262 SRP189094 SRS4518126 GSM3682262 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-HR3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682262 GSM3682262 GEO Accession GSM3682262 SRX5554232 GSM3682263 SRP189094 SRS4518127 GSM3682263 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682263 GSM3682263 GEO Accession GSM3682263 SRX5554233 GSM3682264 SRP189094 SRS4518128 GSM3682264 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682264 GSM3682264 GEO Accession GSM3682264 SRX5554234 GSM3682265 SRP189094 SRS4518129 GSM3682265 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682265 GSM3682265 GEO Accession GSM3682265 SRX5554235 GSM3682266 SRP189094 SRS4518131 GSM3682266 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682266 GSM3682266 GEO Accession GSM3682266 SRX5554236 GSM3682267 SRP189094 SRS4518130 GSM3682267 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682267 GSM3682267 GEO Accession GSM3682267 SRX5554237 GSM3682268 SRP189094 SRS4518132 GSM3682268 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682268 GSM3682268 GEO Accession GSM3682268 SRX5554238 GSM3682269 SRP189094 SRS4518133 GSM3682269 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682269 GSM3682269 GEO Accession GSM3682269 SRX5554239 GSM3682270 SRP189094 SRS4518135 GSM3682270 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682270 GSM3682270 GEO Accession GSM3682270 SRX5554240 GSM3682271 SRP189094 SRS4518134 GSM3682271 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682271 GSM3682271 GEO Accession GSM3682271 SRX5554241 GSM3682272 SRP189094 SRS4518136 GSM3682272 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682272 GSM3682272 GEO Accession GSM3682272 SRX5554242 GSM3682273 SRP189094 SRS4518137 GSM3682273 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682273 GSM3682273 GEO Accession GSM3682273 SRX5554243 GSM3682274 SRP189094 SRS4518138 GSM3682274 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682274 GSM3682274 GEO Accession GSM3682274 SRX5554244 GSM3682275 SRP189094 SRS4518139 GSM3682275 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682275 GSM3682275 GEO Accession GSM3682275 SRX5554245 GSM3682276 SRP189094 SRS4518140 GSM3682276 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682276 GSM3682276 GEO Accession GSM3682276 SRX5554246 GSM3682277 SRP189094 SRS4518141 GSM3682277 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682277 GSM3682277 GEO Accession GSM3682277 SRX5554247 GSM3682278 SRP189094 SRS4518142 GSM3682278 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682278 GSM3682278 GEO Accession GSM3682278 SRX5554248 GSM3682279 SRP189094 SRS4518143 GSM3682279 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682279 GSM3682279 GEO Accession GSM3682279 SRX5554249 GSM3682280 SRP189094 SRS4518144 GSM3682280 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682280 GSM3682280 GEO Accession GSM3682280 SRX5554250 GSM3682281 SRP189094 SRS4518145 GSM3682281 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682281 GSM3682281 GEO Accession GSM3682281 SRX5554251 GSM3682282 SRP189094 SRS4518147 GSM3682282 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682282 GSM3682282 GEO Accession GSM3682282 SRX5554252 GSM3682283 SRP189094 SRS4518146 GSM3682283 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682283 GSM3682283 GEO Accession GSM3682283 SRX5554253 GSM3682284 SRP189094 SRS4518148 GSM3682284 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682284 GSM3682284 GEO Accession GSM3682284 SRX5554254 GSM3682285 SRP189094 SRS4518149 GSM3682285 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682285 GSM3682285 GEO Accession GSM3682285 SRX5554255 GSM3682286 SRP189094 SRS4518150 GSM3682286 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682286 GSM3682286 GEO Accession GSM3682286 SRX5554256 GSM3682287 SRP189094 SRS4518151 GSM3682287 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682287 GSM3682287 GEO Accession GSM3682287 SRX5554257 GSM3682288 SRP189094 SRS4518152 GSM3682288 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682288 GSM3682288 GEO Accession GSM3682288 SRX5554258 GSM3682289 SRP189094 SRS4518153 GSM3682289 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682289 GSM3682289 GEO Accession GSM3682289 SRX5554259 GSM3682290 SRP189094 SRS4518154 GSM3682290 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG-GFP-AtRPL18-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682290 GSM3682290 GEO Accession GSM3682290 SRX5554260 GSM3682291 SRP189094 SRS4518155 GSM3682291 RNA-Seq TRANSCRIPTOMIC cDNA 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) RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682291 GSM3682291 GEO Accession GSM3682291 SRX5554261 GSM3682292 SRP189094 SRS4518156 GSM3682292 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682292 GSM3682292 GEO Accession GSM3682292 SRX5554262 GSM3682293 SRP189094 SRS4518157 GSM3682293 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682293 GSM3682293 GEO Accession GSM3682293 SRX5554263 GSM3682294 SRP189094 SRS4518158 GSM3682294 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682294 GSM3682294 GEO Accession GSM3682294 SRX5554264 GSM3682295 SRP189094 SRS4518159 GSM3682295 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682295 GSM3682295 GEO Accession GSM3682295 SRX5554265 GSM3682296 SRP189094 SRS4518160 GSM3682296 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682296 GSM3682296 GEO Accession GSM3682296 SRX5554266 GSM3682297 SRP189094 SRS4518161 GSM3682297 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682297 GSM3682297 GEO Accession GSM3682297 SRX5554267 GSM3682298 SRP189094 SRS4518162 GSM3682298 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682298 GSM3682298 GEO Accession GSM3682298 SRX5554268 GSM3682299 SRP189094 SRS4518163 GSM3682299 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682299 GSM3682299 GEO Accession GSM3682299 SRX5554269 GSM3682300 SRP189094 SRS4518164 GSM3682300 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682300 GSM3682300 GEO Accession GSM3682300 SRX5554270 GSM3682301 SRP189094 SRS4518165 GSM3682301 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682301 GSM3682301 GEO Accession GSM3682301 SRX5554271 GSM3682302 SRP189094 SRS4518166 GSM3682302 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682302 GSM3682302 GEO Accession GSM3682302 SRX5554272 GSM3682303 SRP189094 SRS4518168 GSM3682303 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682303 GSM3682303 GEO Accession GSM3682303 SRX5554273 GSM3682304 SRP189094 SRS4518167 GSM3682304 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682304 GSM3682304 GEO Accession GSM3682304 SRX5554274 GSM3682305 SRP189094 SRS4518169 GSM3682305 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682305 GSM3682305 GEO Accession GSM3682305 SRX5554275 GSM3682306 SRP189094 SRS4518170 GSM3682306 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682306 GSM3682306 GEO Accession GSM3682306 SRX5554276 GSM3682307 SRP189094 SRS4518171 GSM3682307 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682307 GSM3682307 GEO Accession GSM3682307 SRX5554277 GSM3682308 SRP189094 SRS4518172 GSM3682308 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682308 GSM3682308 GEO Accession GSM3682308 SRX5554278 GSM3682309 SRP189094 SRS4518173 GSM3682309 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682309 GSM3682309 GEO Accession GSM3682309 SRX5554279 GSM3682310 SRP189094 SRS4518175 GSM3682310 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682310 GSM3682310 GEO Accession GSM3682310 SRX5554280 GSM3682311 SRP189094 SRS4518174 GSM3682311 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682311 GSM3682311 GEO Accession GSM3682311 SRX5554281 GSM3682312 SRP189094 SRS4518176 GSM3682312 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682312 GSM3682312 GEO Accession GSM3682312 SRX5554282 GSM3682313 SRP189094 SRS4518177 GSM3682313 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682313 GSM3682313 GEO Accession GSM3682313 SRX5554283 GSM3682314 SRP189094 SRS4518178 GSM3682314 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682314 GSM3682314 GEO Accession GSM3682314 SRX5554284 GSM3682315 SRP189094 SRS4518179 GSM3682315 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682315 GSM3682315 GEO Accession GSM3682315 SRX5554285 GSM3682316 SRP189094 SRS4518180 GSM3682316 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682316 GSM3682316 GEO Accession GSM3682316 SRX5554286 GSM3682317 SRP189094 SRS4518181 GSM3682317 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682317 GSM3682317 GEO Accession GSM3682317 SRX5554287 GSM3682318 SRP189094 SRS4518182 GSM3682318 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682318 GSM3682318 GEO Accession GSM3682318 SRX5554288 GSM3682319 SRP189094 SRS4518183 GSM3682319 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682319 GSM3682319 GEO Accession GSM3682319 SRX5554289 GSM3682320 SRP189094 SRS4518184 GSM3682320 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682320 GSM3682320 GEO Accession GSM3682320 SRX5554290 GSM3682321 SRP189094 SRS4518185 GSM3682321 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682321 GSM3682321 GEO Accession GSM3682321 SRX5554291 GSM3682322 SRP189094 SRS4518186 GSM3682322 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682322 GSM3682322 GEO Accession GSM3682322 SRX5554292 GSM3682323 SRP189094 SRS4518187 GSM3682323 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682323 GSM3682323 GEO Accession GSM3682323 SRX5554293 GSM3682324 SRP189094 SRS4518188 GSM3682324 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682324 GSM3682324 GEO Accession GSM3682324 SRX5554294 GSM3682325 SRP189094 SRS4518189 GSM3682325 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682325 GSM3682325 GEO Accession GSM3682325 SRX5554295 GSM3682326 SRP189094 SRS4518190 GSM3682326 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682326 GSM3682326 GEO Accession GSM3682326 SRX5554296 GSM3682327 SRP189094 SRS4518191 GSM3682327 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682327 GSM3682327 GEO Accession GSM3682327 SRX5554297 GSM3682328 SRP189094 SRS4518192 GSM3682328 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682328 GSM3682328 GEO Accession GSM3682328 SRX5554298 GSM3682329 SRP189094 SRS4518193 GSM3682329 RNA-Seq TRANSCRIPTOMIC cDNA Nuclei were purified from frozen and pulverized tissue as described previously for Arabidopsis thaliana (Wang and Deal, 2015) with only minor modifications including 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 (Celltrics) 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 (this suspension is considered input of the purification), Twenty-five microliters of M-280 streptavidin-coated Dynabeads (~1.5 x 107 beads; Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated to allow capture of the nuclei 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 7 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 7 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube. For RNA extraction, the bead-bound nuclei mixture was placed on a 2 mL tube magnet for 4 min at 4° C to capture the nuclei, which were resuspended in 20 µl NPB before proceeding to the extraction For nuclear RNA libraries, rRNA was degraded with probes that were hybridized with up to 1 µg RNA in a 6 μl reaction containing 0.1 μM of each probe and 1 μl of 5x hybridization buffer (0.5 M Tris-HCl [pH 7.0], 1 M NaCl). The probe concentration was scaled down proportionally for lower amounts of RNA. For denaturation and hybridization, the sample was heated to 95 °C for 2 min, cooled to 45 °C at 0.1 °C/s and incubated at 45 °C for 5 min. For RNase treatment, the hybridization reaction at 45 °C was increased to 10 μl by adding 5 U of thermostable RNase H Hybridase (Epicentre) and 1 μl of Hybridase buffer (500 mM Tris-HCl [pH 7.4], 1 M NaCl, 200 mM MgCl2), and incubated at 45 °C for 30 min. To remove the DNA probes and degraded RNA from the samples, the sample was re-treated with Turbo DNase I (ThermoFisher Scientific) and cleaned up with Agencourt RNAClean XP beads (Beckman Coulter). RNA-seq libraries of pre/rRNA degraded nuclear were prepared as described by 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 Illumina HiSeq 3000 gds 303682329 GSM3682329 GEO Accession GSM3682329 SRX5554299 GSM3682330 SRP189094 SRS4518194 GSM3682330 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6-FLAG-GFP-AtRPL18B-1 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682330 GSM3682330 GEO Accession GSM3682330 SRX5554300 GSM3682331 SRP189094 SRS4518195 GSM3682331 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6-FLAG-GFP-AtRPL18-5 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 Illumina HiSeq 3000 gds 303682331 GSM3682331 GEO Accession GSM3682331 SRX5554301 GSM3682332 SRP189094 SRS4518196 GSM3682332 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682332 GSM3682332 GEO Accession GSM3682332 SRX5554302 GSM3682333 SRP189094 SRS4518197 GSM3682333 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682333 GSM3682333 GEO Accession GSM3682333 SRX5554303 GSM3682334 SRP189094 SRS4518198 GSM3682334 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682334 GSM3682334 GEO Accession GSM3682334 SRX5554304 GSM3682335 SRP189094 SRS4518199 GSM3682335 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682335 GSM3682335 GEO Accession GSM3682335 SRX5554305 GSM3682336 SRP189094 SRS4518201 GSM3682336 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682336 GSM3682336 GEO Accession GSM3682336 SRX5554306 GSM3682337 SRP189094 SRS4518200 GSM3682337 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682337 GSM3682337 GEO Accession GSM3682337 SRX5554307 GSM3682338 SRP189094 SRS4518203 GSM3682338 OTHER TRANSCRIPTOMIC other 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682338 GSM3682338 GEO Accession GSM3682338 SRX5554308 GSM3682339 SRP189094 SRS4518202 GSM3682339 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682339 GSM3682339 GEO Accession GSM3682339 SRX5554309 GSM3682340 SRP189094 SRS4518206 GSM3682340 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682340 GSM3682340 GEO Accession GSM3682340 SRX5554310 GSM3682341 SRP189094 SRS4518204 GSM3682341 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682341 GSM3682341 GEO Accession GSM3682341 SRX5554311 GSM3682342 SRP189094 SRS4518205 GSM3682342 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682342 GSM3682342 GEO Accession GSM3682342 SRX5554312 GSM3682343 SRP189094 SRS4518207 GSM3682343 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682343 GSM3682343 GEO Accession GSM3682343 SRX5554313 GSM3682344 SRP189094 SRS4518208 GSM3682344 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682344 GSM3682344 GEO Accession GSM3682344 SRX5554314 GSM3682345 SRP189094 SRS4518209 GSM3682345 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682345 GSM3682345 GEO Accession GSM3682345 SRX5554315 GSM3682346 SRP189094 SRS4518210 GSM3682346 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682346 GSM3682346 GEO Accession GSM3682346 SRX5554317 GSM3682348 SRP189094 SRS4518212 GSM3682348 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682348 GSM3682348 GEO Accession GSM3682348 SRX5554318 GSM3682349 SRP189094 SRS4518213 GSM3682349 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682349 GSM3682349 GEO Accession GSM3682349 SRX5554319 GSM3682350 SRP189094 SRS4518214 GSM3682350 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682350 GSM3682350 GEO Accession GSM3682350 SRX5554321 GSM3682352 SRP189094 SRS4518217 GSM3682352 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682352 GSM3682352 GEO Accession GSM3682352 SRX5554322 GSM3682353 SRP189094 SRS4518216 GSM3682353 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682353 GSM3682353 GEO Accession GSM3682353 SRX5554323 GSM3682354 SRP189094 SRS4518218 GSM3682354 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682354 GSM3682354 GEO Accession GSM3682354 SRX5554324 GSM3682355 SRP189094 SRS4518219 GSM3682355 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682355 GSM3682355 GEO Accession GSM3682355 SRX5554327 GSM3682358 SRP189094 SRS4518222 GSM3682358 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682358 GSM3682358 GEO Accession GSM3682358 SRX5554329 GSM3682360 SRP189094 SRS4518224 GSM3682360 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682360 GSM3682360 GEO Accession GSM3682360 SRX5554330 GSM3682361 SRP189094 SRS4518225 GSM3682361 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682361 GSM3682361 GEO Accession GSM3682361 SRX5554332 GSM3682363 SRP189094 SRS4518229 GSM3682363 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682363 GSM3682363 GEO Accession GSM3682363 SRX5554333 GSM3682364 SRP189094 SRS4518228 GSM3682364 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682364 GSM3682364 GEO Accession GSM3682364 SRX5554334 GSM3682365 SRP189094 SRS4518227 GSM3682365 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682365 GSM3682365 GEO Accession GSM3682365 SRX5554335 GSM3682366 SRP189094 SRS4518230 GSM3682366 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682366 GSM3682366 GEO Accession GSM3682366 SRX5554336 GSM3682367 SRP189094 SRS4518231 GSM3682367 OTHER GENOMIC other Genomic DNA was isolated using the Dneasy Plant MiniKit (Qiagen) Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using isolated DNA as previously described (Maher et al 2017, Bajic et al 2018) 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 NextSeq 500 gds 303682367 GSM3682367 GEO Accession GSM3682367 SRX5554337 GSM3682368 SRP189094 SRS4518232 GSM3682368 OTHER GENOMIC other Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using isolated DNA as previously described (Maher et al 2017, Bajic et al 2018) Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using isolated DNA as previously described (Maher et al 2017, Bajic et al 2018) 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 NextSeq 500 gds 303682368 GSM3682368 GEO Accession GSM3682368 SRX5554338 GSM3682369 SRP189094 SRS4518233 GSM3682369 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682369 GSM3682369 GEO Accession GSM3682369 SRX5554339 GSM3682370 SRP189094 SRS4518234 GSM3682370 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682370 GSM3682370 GEO Accession GSM3682370 SRX5554340 GSM3682371 SRP189094 SRS4518235 GSM3682371 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682371 GSM3682371 GEO Accession GSM3682371 SRX5554341 GSM3682372 SRP189094 SRS4518236 GSM3682372 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682372 GSM3682372 GEO Accession GSM3682372 SRX5554342 GSM3682373 SRP189094 SRS4518237 GSM3682373 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682373 GSM3682373 GEO Accession GSM3682373 SRX5554343 GSM3682374 SRP189094 SRS4518238 GSM3682374 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682374 GSM3682374 GEO Accession GSM3682374 SRX5554344 GSM3682375 SRP189094 SRS4518239 GSM3682375 ATAC-seq GENOMIC other Genomic DNA was isolated using the Dneasy Plant MiniKit (Qiagen) Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using isolated DNA as previously described (Maher et al 2017, Bajic et al 2018) 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 NextSeq 500 gds 303682375 GSM3682375 GEO Accession GSM3682375 SRX5554345 GSM3682376 SRP189094 SRS4518240 GSM3682376 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682376 GSM3682376 GEO Accession GSM3682376 SRX5554346 GSM3682377 SRP189094 SRS4518241 GSM3682377 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682377 GSM3682377 GEO Accession GSM3682377 SRX5554347 GSM3682378 SRP189094 SRS4518242 GSM3682378 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682378 GSM3682378 GEO Accession GSM3682378 SRX5554348 GSM3682379 SRP189094 SRS4518243 GSM3682379 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682379 GSM3682379 GEO Accession GSM3682379 SRX5554349 GSM3682380 SRP189094 SRS4518244 GSM3682380 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682380 GSM3682380 GEO Accession GSM3682380 SRX5554350 GSM3682381 SRP189094 SRS4518245 GSM3682381 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682381 GSM3682381 GEO Accession GSM3682381 SRX5554351 GSM3682382 SRP189094 SRS4518246 GSM3682382 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682382 GSM3682382 GEO Accession GSM3682382 SRX5554352 GSM3682383 SRP189094 SRS4518247 GSM3682383 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 NextSeq 500 gds 303682383 GSM3682383 GEO Accession GSM3682383 SRX5554353 GSM3682384 SRP189094 SRS4518248 GSM3682384 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682384 GSM3682384 GEO Accession GSM3682384 SRX5554354 GSM3682385 SRP189094 SRS4518249 GSM3682385 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682385 GSM3682385 GEO Accession GSM3682385 SRX5554355 GSM3682386 SRP189094 SRS4518250 GSM3682386 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682386 GSM3682386 GEO Accession GSM3682386 SRX5554356 GSM3682387 SRP189094 SRS4518251 GSM3682387 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682387 GSM3682387 GEO Accession GSM3682387 SRX5554357 GSM3682388 SRP189094 SRS4518252 GSM3682388 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682388 GSM3682388 GEO Accession GSM3682388 SRX5554358 GSM3682389 SRP189094 SRS4518253 GSM3682389 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682389 GSM3682389 GEO Accession GSM3682389 SRX5554359 GSM3682390 SRP189094 SRS4518255 GSM3682390 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682390 GSM3682390 GEO Accession GSM3682390 SRX5554360 GSM3682391 SRP189094 SRS4518254 GSM3682391 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, p35S:His6-FLAG-GFP-MtRPL18-3 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 RNA-seq libraries were prepared from poly(A)+ selected mRNA and described by Townsley et al. (2014) for random primer primed libraries. 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 NextSeq 500 gds 303682391 GSM3682391 GEO Accession GSM3682391 SRX5554361 GSM3682392 SRP189094 SRS4518256 GSM3682392 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682392 GSM3682392 GEO Accession GSM3682392 SRX5554362 GSM3682393 SRP189094 SRS4518257 GSM3682393 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682393 GSM3682393 GEO Accession GSM3682393 SRX5554363 GSM3682394 SRP189094 SRS4518258 GSM3682394 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682394 GSM3682394 GEO Accession GSM3682394 SRX5554364 GSM3682395 SRP189094 SRS4518259 GSM3682395 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682395 GSM3682395 GEO Accession GSM3682395 SRX5554365 GSM3682396 SRP189094 SRS4518260 GSM3682396 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682396 GSM3682396 GEO Accession GSM3682396 SRX5554366 GSM3682397 SRP189094 SRS4518261 GSM3682397 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682397 GSM3682397 GEO Accession GSM3682397 SRX5554367 GSM3682398 SRP189094 SRS4518262 GSM3682398 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682398 GSM3682398 GEO Accession GSM3682398 SRX5554368 GSM3682399 SRP189094 SRS4518263 GSM3682399 RNA-Seq TRANSCRIPTOMIC cDNA 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 NextSeq 500 gds 303682399 GSM3682399 GEO Accession GSM3682399 SRX5554369 GSM3682400 SRP189094 SRS4518264 GSM3682400 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682400 GSM3682400 GEO Accession GSM3682400 SRX5554370 GSM3682401 SRP189094 SRS4518265 GSM3682401 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682401 GSM3682401 GEO Accession GSM3682401 SRX5554371 GSM3682402 SRP189094 SRS4518266 GSM3682402 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682402 GSM3682402 GEO Accession GSM3682402 SRX5554372 GSM3682403 SRP189094 SRS4518268 GSM3682403 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682403 GSM3682403 GEO Accession GSM3682403 SRX5554373 GSM3682404 SRP189094 SRS4518267 GSM3682404 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682404 GSM3682404 GEO Accession GSM3682404 SRX5554374 GSM3682405 SRP189094 SRS4518269 GSM3682405 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682405 GSM3682405 GEO Accession GSM3682405 SRX5554375 GSM3682406 SRP189094 SRS4518270 GSM3682406 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682406 GSM3682406 GEO Accession GSM3682406 SRX5554376 GSM3682407 SRP189094 SRS4518271 GSM3682407 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682407 GSM3682407 GEO Accession GSM3682407 SRX5554377 GSM3682408 SRP189094 SRS4518272 GSM3682408 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682408 GSM3682408 GEO Accession GSM3682408 SRX5554378 GSM3682409 SRP189094 SRS4518273 GSM3682409 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682409 GSM3682409 GEO Accession GSM3682409 SRX5554379 GSM3682410 SRP189094 SRS4518275 GSM3682410 ATAC-seq GENOMIC other 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) 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 NextSeq 500 gds 303682410 GSM3682410 GEO Accession GSM3682410 SRX5554380 GSM3682411 SRP189094 SRS4518274 GSM3682411 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682411 GSM3682411 GEO Accession GSM3682411 SRX5554381 GSM3682412 SRP189094 SRS4518276 GSM3682412 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682412 GSM3682412 GEO Accession GSM3682412 SRX5554382 GSM3682413 SRP189094 SRS4518277 GSM3682413 ATAC-seq GENOMIC other 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) 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 Illumina HiSeq 3000 gds 303682413 GSM3682413 GEO Accession GSM3682413 SRX5554383 GSM3682414 SRP189094 SRS4518278 GSM3682414 ATAC-seq GENOMIC other Genomic DNA was isolated using the Dneasy Plant MiniKit (Qiagen) Tagmentation using Tn5 insertion and ATAC-seq libraries were prepared using isolated DNA as previously described (Maher et al 2017, Bajic et al 2018) 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 NextSeq 500 gds 303682414 GSM3682414 GEO Accession GSM3682414 SRX5554384 GSM3682415 SRP189094 SRS4518279 GSM3682415 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682415 GSM3682415 GEO Accession GSM3682415 SRX5554385 GSM3682416 SRP189094 SRS4518280 GSM3682416 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682416 GSM3682416 GEO Accession GSM3682416 SRX5554386 GSM3682417 SRP189094 SRS4518281 GSM3682417 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682417 GSM3682417 GEO Accession GSM3682417 SRX5554387 GSM3682418 SRP189094 SRS4518282 GSM3682418 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682418 GSM3682418 GEO Accession GSM3682418 SRX5554388 GSM3682419 SRP189094 SRS4518283 GSM3682419 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682419 GSM3682419 GEO Accession GSM3682419 SRX5554389 GSM3682420 SRP189094 SRS4518284 GSM3682420 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682420 GSM3682420 GEO Accession GSM3682420 SRX5554390 GSM3682421 SRP189094 SRS4518285 GSM3682421 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682421 GSM3682421 GEO Accession GSM3682421 SRX5554391 GSM3682422 SRP189094 SRS4518286 GSM3682422 RNA-Seq TRANSCRIPTOMIC cDNA 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 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 Illumina HiSeq 3000 gds 303682422 GSM3682422 GEO Accession GSM3682422 SRX5554392 GSM3682423 SRP189094 SRS4518287 GSM3682423 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682423 GSM3682423 GEO Accession GSM3682423 SRX5554393 GSM3682424 SRP189094 SRS4518288 GSM3682424 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682424 GSM3682424 GEO Accession GSM3682424 SRX5554394 GSM3682425 SRP189094 SRS4518289 GSM3682425 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682425 GSM3682425 GEO Accession GSM3682425 SRX5554395 GSM3682426 SRP189094 SRS4518291 GSM3682426 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682426 GSM3682426 GEO Accession GSM3682426 SRX5554396 GSM3682427 SRP189094 SRS4518292 GSM3682427 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682427 GSM3682427 GEO Accession GSM3682427 SRX5554397 GSM3682428 SRP189094 SRS4518290 GSM3682428 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682428 GSM3682428 GEO Accession GSM3682428 SRX5554398 GSM3682429 SRP189094 SRS4518293 GSM3682429 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682429 GSM3682429 GEO Accession GSM3682429 SRX5554399 GSM3682430 SRP189094 SRS4518295 GSM3682430 RNA-Seq TRANSCRIPTOMIC cDNA For cellular mRNA, 35S:His6:FLAG:OsRPL18-2 pulverized tissue was extracted in PEB and a S16 fraction was obtained as detailed by Mustroph et al. 2009. Total poly(A)+ RNA was extracted by direct capture of poly(A)+ RNA using a biotinylated oligo dT according to Townsley et al., 2015 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 Illumina HiSeq 3000 gds 303682430 GSM3682430 GEO Accession GSM3682430 SRX5554400 GSM3682431 SRP189094 SRS4518294 GSM3682431 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682431 GSM3682431 GEO Accession GSM3682431 SRX5554401 GSM3682432 SRP189094 SRS4518296 GSM3682432 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682432 GSM3682432 GEO Accession GSM3682432 SRX5554402 GSM3682433 SRP189094 SRS4518297 GSM3682433 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682433 GSM3682433 GEO Accession GSM3682433 SRX5554403 GSM3682434 SRP189094 SRS4518298 GSM3682434 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682434 GSM3682434 GEO Accession GSM3682434 SRX5554404 GSM3682435 SRP189094 SRS4518299 GSM3682435 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682435 GSM3682435 GEO Accession GSM3682435 SRX5554405 GSM3682436 SRP189094 SRS4518301 GSM3682436 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682436 GSM3682436 GEO Accession GSM3682436 SRX5554406 GSM3682437 SRP189094 SRS4518300 GSM3682437 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682437 GSM3682437 GEO Accession GSM3682437 SRX5554407 GSM3682438 SRP189094 SRS4518302 GSM3682438 RNA-Seq TRANSCRIPTOMIC cDNA 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 Illumina HiSeq 3000 gds 303682438 GSM3682438 GEO Accession GSM3682438 SRX5554408 GSM3682439 SRP189094 SRS4518303 GSM3682439 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682439 GSM3682439 GEO Accession GSM3682439 SRX5554409 GSM3682440 SRP189094 SRS4518304 GSM3682440 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682440 GSM3682440 GEO Accession GSM3682440 SRX5554410 GSM3682441 SRP189094 SRS4518305 GSM3682441 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682441 GSM3682441 GEO Accession GSM3682441 SRX5554411 GSM3682442 SRP189094 SRS4518306 GSM3682442 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682442 GSM3682442 GEO Accession GSM3682442 SRX5554412 GSM3682443 SRP189094 SRS4518307 GSM3682443 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682443 GSM3682443 GEO Accession GSM3682443 SRX5554413 GSM3682444 SRP189094 SRS4518308 GSM3682444 RNA-Seq TRANSCRIPTOMIC cDNA 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) 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 . Library construction continued as described by Bazin et al 2017, and the resultant 130 nt RF cDNAs were circularized and contaminating rRNA was subtracted by a second hybridization with custom-designed biotinylated oligos corresponding to pre-rRNA and rRNA as described (14). rRNA-subtracted circularized fragments were used for 12 cycles of 10s at 98°C, 10s at 60°C, and 5s at 72 °C PCR amplification including library and indexing primers 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 Illumina HiSeq 3000 gds 303682444 GSM3682444 GEO Accession GSM3682444