title: TOTCONSPSHOOTC [C03_TOTAL_repeat]

organism: Solanum pennellii

extract_protocol:
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

value: Solanum pennellii LA0716

value: Ribosome footprints


title: ChIP_Brachypodium_7days_leaf_H3K4me3

status: Public on Sep 19 2019

type: SRA

city: Athens

first: Robert

middle: J

last: Schmitz

state: GA

address: B416 Davison Life Sciences

department: Genetics

country: USA

institute: University of Georgia

library_source: transcriptomic

data_processing:
RNA-seq: analysis was performed using Tophat v2.1.1 and cufflinks v2.2.1
ChIP-seq: analysis was performed using bowtie1.1.1
ATAC-seq:analysis was performed using bowtie1.1.1, macs2
Genome_build: Arabidopsis thaliana, Eutrema salsugineum, Phaseolus vulgaris,  Glycine max, Brachypodium distachyon, Oryza sativa, Setaria viridis, Populus trichocarpa, Sorghum bicolor genomes were from JGI (phytozome version 11) ; Zea mays from maizeGDB AGP_v4; Hordeum vulgare from ensemble (version 42) ; Asparagus officinalis from Asparagus genome project (http://asparagus.uga.edu/tripal/);  Spirodela polyrhiza from Michael et al..
Supplementary_files_format_and_content: *BED file, the identified regulatory regions, tab delimited text file, see ACR_column_header.txt for column headers
Supplementary_files_format_and_content: *BIGWIG , the genome coverage of each sample

molecule: total RNA

organism: Brachypodium distachyon

source_name: DNA

growth_protocol: Arabidopsis thaliana (accession Col-0) were grown on 1/2MS plates under 25°C and continues light for 7 days. Leaves were collected for the experiments. Spirodela polyrhiza (accession 7498) were grown in liquid bases (Schenk and Hilldebrandt basak salt mixture) under 25°C and continuous light. Whole seedlings were collected 3 days after transferring to a new basis. Eutrema salsugineum (accession Shandong) and Asparagus officinalis (accession Gijnlim) were grown in the soil for approximately 10 days under 25°C and photoperiodic lighting (16 hours of light:8 hours of dark). All the young leaves were collected for the experiments. Phaseolus vulgaris (accession G19833) and Glycine max (accession William82) were grown in the soil for approximately 10 days under 25°C and photoperiodic lighting (16 hours of light:8 hours of dark). The 2nd and 3rd leaves were collected for the experiments. Brachypodium distachyon (accession Bd21), Oryza sativa (accession Nipponbare), Setaria viridis (accession A10), Sorghum bicolor, Zea mays (accession B73), and Hordeum vulgare (accession Morex) were grown in soil for ~6-7days under 25°C, 16 hours of light/8 hours of dark. The inner second leaves—which contains the third and fourth leaves sheathed inside—were used for experiments. Populus trichocarpa leaves were collected from the tree and flash-frozen with liquid N2 immediately for the following experiments.

extract_protocol: ChIP-seq: ChiP-seq was performed following the methods of Zhang et al (2007) (https://www.ncbi.nlm.nih.gov/pubmed/17439305).  Tissues were cross-linked with 1% formaldehyde and chromatin was sonicated, followed by immunoprecipitation.

tag: accession

tag: tissue

value: leaf

tag: age


title: TRACONSLSHOOTB [B01_TRAP]

city: Atlanta

first: Roger

middle: B

last: Deal

address: 1510 Clifton Rd NE

department: Biology

institute: Emory University

data_processing:
Genome_build: Genome_build:  RICE: IRGSP-1.0-30 (https://rapdb.dna.affrc.go.jp/download/irgsp1.html), MEDICAGO: Mt4.0 (http://www.medicagogenome.org/downloads), SOLANUM LYCOPERSICUM: ITAG3.10 (ftp://ftp.solgenomics.net/tomato_genome/assembly/build_3.00/), SOLANUM PENNELLII: Spenn-ENA-HG975439-HG975452 (https://www.ebi.ac.uk/ena/data/view/HG975439-HG975452)
Rice and Medicago RNA-seq raw reads were aligned to the IGRSP-1.0-30 and Mt4.0 genome, respectively, with the splice junction aware short read alignment suite Bowtie2/Tophat2, allowing unique alignments with 2 or less mismatches
Rice and Medicago nRNA, polyadenylated total RNA, and polyadenylated TRAP RNA filtering was done by first aligning the reads to mitochondrial and chloroplast genomes before mapping to the nuclear genome
Rice and Medicago read count data for RNA-seq was generated for features of exons-by-genes using the summarize Overlaps function of the GenomicRanges package.
Solanum RNA-seq mapping was performed in the CyVerse Discovery Environment
Solanum nRNA-seq reads were filtered, using STAR v2.4.0.1, by first aligning to organelle sequences (S. lycoperscium AFYB01.1 mitochondrial sequence, S. lycopersicum NC_007898.3 chloroplast sequence, and S. pennellii HG74452 chloroplast sequence) and then to either S. lycopersicum ITAG3.10 or S. pennellii exonic sequences
Solanum read count data for RNA-seq was generated the est_count feature of eXpress v1.5.1
ATAC-seq raw reads were aligned to each species' corresponding genome using Bowtie2 with default parameters
ATAC-seq mapped reads were filtered to remove 1) reads that mapped to organelle genomes and 2) reads with a mapping quality score of q<2.
Transposase Hypersensitive Sites were identified using the Findpeaks function of the HOMER package, with the parameters "-minDist 150" "-region" and "-regionRes 1"
Filtered ATAC-seq bam files for each sample were converted to the bigwig format using a bin size resolution of 1 base pair and the "--normalizeUsingRPKM" option
For accurate visualization, all filtered ATAC-seq bam files for a given tissue were scaled to the same number of reads and combined per condition to create a single merged bam file for a condition in a tissue type. The merged bam file was converted to a bigwig file using a bin size resolution of 1 base pair and the "--normalizeUsingRPKM" option
Filtered ATAC-seq bam files from genomic DNA samples were scaled to the total number of reads present in the merged bam files for a given tissue type. The scaled bam file was converted to a bigwig file using a bin size resolution of 1 base pair and the "--normalizeUsingRPKM" option
Supplementary_files_format_and_content: Reads per kilobase per million reads (RPKM) values for nRNA, polyA RNA or TRAP RNA datasets were calculated from count data after quantile normalization
Supplementary_files_format_and_content: .bed files contain Transposase Hypersensitive Site (THS) coordinates for individual samples or for each tissue type they are organized as (All) all the replicated THSs identified in that sample set in either control or submergence samples, (Up) THSs upregulated in submergence samples, and (Down) THSs downregulated in submergence samples
Supplementary_files_format_and_content: .bw files contain aligned reads for individual samples, or combined samples for a specific condition of a given tissue, at a resolution of 1 bp bin sized windows

organism: Solanum lycopersicum

source_name: 7 days-old seedling shoot apical meristem

growth_protocol: For rice, seeds were dehulled and surface sterilized in 50% (v/v) bleach solution for 30 min, rinsed ten times with sterile distilled water and grown on plates (100 cm2) containing 0.5x Murashige and Skoog medium (MS), 1% (w/v) agar 1% (w/v) sucrose for 7 days (16h day / 8h night; at 28°C/25°C day/night; 110 μEm-2s-1). For tomato, seeds were surface sterilized in 50% (v/v) bleach solution for 5 min (S. pennellii) or 20 min (S. lycopersicum) and then rinsed three times with sterile distilled water. Growth was on vertical plates (10 cm x 10 cm) containing full-strength MS without vitamins, with 1% (w/v) agar (w/v) and 1% (w/v) sucrose.  Hairy root cultures of Agrobacterium rhizogenes transformed S. lycopersicum were initiated and cultivated as described (Ron et al. 2014). Cultures were subcloned using a 2-cm hairy root segment, grown on horizontal plates (10 cm x 10 cm) containing full-strength MS with vitamins, with 1% (w/v) agar (w/v) and 3% (w/v) sucrose, 200 mg/L kanamycin and 200 mg/L cefotaxime. All tomato root cultures or germinating seeds were grown for 7 days in a growth chamber (at 25°C, 16h day/8h night; 60-65 μEm-2s-1). M. truncatula seeds were surface-sterilized by incubating in concentrated sulfuric acid for 8 minutes with gentle stirring, washing 3 times with 4°C sterile, distilled water, then 4-8 minutes in 3% (w/v) hypochlorite (diluted bleach), washing 4 times with sterile, distilled water, and finally placing the seeds onto moist filter paper. Seeds were germinated without vernalization on moist filter papers in inverted Petri dishes wrapped with surgical tape wrapping. These were kept in the growth room in the dark at 20°C for 2 days. Hairy root composite M. truncatula plants were initiated by injecting with A. rhizogenes K599. To do so, the day before injection, a volume of fresh overnight culture of A. rhizogenes K599 carrying either 35S:NTF;AtACT2p:mBirA or 35S:His6-FLAG-GFP-MtRPL18-3 plasmid was used to inoculate  5 ml of Yeast Extract Beef media (5 g/L tryptone, 1 g/L yeast extract, 5 g/L nutrient broth, 5 g/L sucrose, 0.49 g/L MgSO4x7H2O, 15 g/L agar, pH=7.2) additionally containing 100 mg/L Spectinomycin and grown overnight at 28°C at 200 rpm. When the OD600 was equal to 1.0, cultures were centrifuged at 5000 rpm for 5 min. The supernatant was poured off and the pellet was resuspended in Injection Media (IM) (1X PBS, 100 uM acetosyringone, 1/10000 (v/v) Silwet). Seedlings germinating for 2.5-days were injected by placing them in 5 mL of IM-resuspended A. rhizogenes poured out on a Petri dish and stabbing the root a few times with an 18G1 needle. Additionally, 1-2 mm of the primary root tip was cut off. Injected seedlings were moved to slanted Fahräeus Media (FM) plates (0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 50 nM FeEDTA, 0.5 mM NH4NO3, 1mM CaCl2, 0.1 mg of MnSO4, CuSO4, Zn SO4, H3BO3, and Na2MoO4, 8 g/L Phytoblend agar, pH 6.5) with no selection and were grown horizontally for 3 days, and then were moved to FM plates with 5 mg/L phosphinothricin and were grown vertically for 3 wk before transfer to 1X MS media without vitamins (1% w/v agar, 1% w/v sucrose). Plants were grown vertically for 1 wk before being used for the submergence experiment. The day before the experiment was performed, root tips from one plate were collected and visualized on a fluorescence stereomicroscope to check for GFP expression. Transformation efficiency was calculated as the percentage of root tips with ubiquitous GFP expression

extract_protocol:
TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (Townsley et al 2014)
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

treatment_protocol:
Transgenic Solanum seeds from transgenic lines were surface sterilized in 50% (v/v) bleach solution for 5 min (S. pennellii) or 20 min (S. lycopersicum) and then rinsed three times with sterile distilled water. Seedlings were grown on plates (10 cm x 10 cm) containing full-strength Murashige and Skoog standard medium (MS) agar (1% w/v) and 1% w/v sucrose, during 7 days in a growth chamber (16 h day / 8 h night;  at 22°C/22°C day/night; 60-65 μEm-2s-1). For submergence treatment ("SUB"), plates were opened and placed horizontally under 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm) and shoot apical meristems were harvested at ZT 6h (2h before relative noon). Control plates ("CON") were not opened, but positioned similarly horizontally and also harvested at ZT 6h.
Four independent biological replicates were grown for each species. For whole plant submergence, plates were placed horizontally, opened and the seedlings covered with 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm; all four species) and the shoot apical meristem region (Solanum species) were harvested at ZT 6h (2h before relative noon). Control plates were not opened, but positioned horizontally for the duration of the treatment and harvested at ZT 6h. Oxygen partial pressure was measured with the NeoFox Sport O2 sensor and probe (Ocean Optics). The dissolved oxygen content in the water covering the plants remained above 18% (v/v) for the 2 h duration of the stress treatment

tag: cultivar

value: Solanum lycopersicum cv. M82

tag: line

tag: experiment type

value: Random primed libraries

tag: molecule subtype

value: TRAP RNA


title: INRNASUBSPSHOOTD2 [IN_D22]

extract_protocol:
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

value: 7 days-old seedling shoot apical meristem

value: nuclear RNA


title: TRASUBSLSHOOTB [B02_TRAP]


title: INRNASUBSPROOTD1 [IN_D16]

source_name: 7 days-old seedling 1 cm roots tips

value: 7 days-old seedling 1 cm roots tips


title: ChIP_Brachypodium_7days_leaf_H3K56ac


title: ChIP_Asparagus_10days_leaf_H3K36me3

organism: Asparagus officinalis

value: Gijnlim


title: INRNACONSPROOTD [IN_D15]


title: ChIP_Brachypodium_7days_leaf_H3K36me3


title: ChIP_Asparagus_10days_leaf_H3K56ac


title: HiC_Sis_log_rep2

city: Kyoto

first: Naomichi

last: Takemata

address: Kyoto University, Katsura, Nishikyo-ku

department: Synthetic Chemistry and Biological Chemistry

country: Japan

institute: Kyoto University

library_source: genomic

data_processing:
Hi-C reads were mapped to the genome of S. acidocaldarius DSM639 (GenBank ID: CP000077.1) or S. islandicus REY15A (GenBank ID: CP002425.1) using HiC-Pro version 2.9.0 (Servant et al., 2015) with default parameters. The genomic coordinates of each Sulfolobus species were re-defined so that they started from the first base of the HindIII site in the original definition. For the overexpression experiment of ClsN, reads were also mapped to the sequence of pSSRgD-ClsN. Valid read were used to generate raw Hi-C matrices. The genome was binned at 15-kb for S. acidocaldarius and 30-kb for S. islandicus. The intra-bin ligations were discarded by setting the values on the corresponding diagonal to zero. The data were iteratively corrected with MAX_ITER = 500 and EPS = 0.1.
ChIP DNA and input DNA were mapped to the genome of S. acidocaldarius DSM639 or S. islandicus REY15A using Bowtie 2 version 2.3.2 with default parameters (Langmead and Salzberg, 2012). Multi-mapping reads were retained for downstream analyses. For a plasmid-containing strain of S. islandicus, reads were separately mapped to the genome or the corresponding plasmid. Read coverage on the chromosome was normalized with the number of reads mapped to the chromosome. To normalize read coverage on the plasmid, numbers of reads mapped to the chromosome and the plasmid were summed and used for read number normalization.
To calculate Transcripts Per Million (TPM), RNA-seq reads from each biological replicate were mapped to protein-coding genes in a strand-specific manner using Salmon version 0.8.2 (Patro et al., 2017).
For marker frequency analysis, reads were mapped to the S. isladicus REY15A genome as described previously (Samson et al., 2013). Read counts were grouped in 1-kb bins.
Genome_build: CP000077.1, CP002425.1
Supplementary_files_format_and_content: Hi-C: tab-delimited tables. The first and second columns show genomic bins. In plasmid-containing strains, bin 86 refers to a corresponding plasmid. The third column shows normalized interaction scores between bins in the first and second columns. ChIP-seq: tab-delimited tables. The first column shows the name of a chromosome or a plasmid. The second column shows coordinates on the chromosome or the plasmid. The third column shows normalized read coverages. RNA-seq: tab-delimited tables showing Transcripts Per Million (TPM) values for each protein-coding genes. Marker frequency analysis: tab-delimited tables showing read counts for each 1-kb bin.

molecule: genomic DNA

organism: Saccharolobus islandicus

source_name: Microorganism

extract_protocol:
Cells were crosslinked, permeabilized, and treated with HindIII to digest cellular DNA. The DNA was end-labeled with biotin and subjected to priximity ligation. After reversal of crosslinks, the DNA was extracted with phenol:chloroform:isoamyl alcohol, precipitated in isopropanol, and sonicated.
Libraries were constructed using NEBNext Ultra DNA Library Prep Kit for Illumina. Biotin-labeled DNA was purified with streptavidin before PCR. Sequencing was performed on the Illumina NextSeq 500 platform.

tag: strain

tag: genotype

value: DpyrEF DlacS

tag: growth phase

value: Log-phase cells


title: ChIP_Asparagus_10days_leaf_H3K27me3


title: ChIP_Brachypodium_7days_leaf_H3K27me3


title: INRNASUBSPSHOOTD1 [IN_D08]


title: HiC_Sis_log_rep1


title: TOTSUBHAIRYROOTT6A [A18_TOTAL]

source_name: hairy root culture 1cm root tips

extract_protocol:
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

treatment_protocol:
Hairy root lines were generated as described (Ron et al. 2014) and grown on horizontal plates (10 cm x 10 cm) containing full-strength MS with vitamins, with agar (1% w/v) and 3% w/v sucrose, 200 mg/L kanamycin and 200 mg/L cefotaxime. Subcloned roots were grown horizontally for 7 days in a growth chamber (16 h day / 8 h night;  at 22°C/22°C day/night; 60-65 μEm-2s-1) prior to the harvest. For submergence treatment ("SUB"), plates were opened and placed horizontally under 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm) were harvested at ZT 6h (2h before relative noon). Control plates ("CON") were not opened, but positioned similarly horizontally and also harvested at ZT 6h.
Four independent biological replicates were grown for each species. For whole plant submergence, plates were placed horizontally, opened and the seedlings covered with 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm; all four species) and the shoot apical meristem region (Solanum species) were harvested at ZT 6h (2h before relative noon). Control plates were not opened, but positioned horizontally for the duration of the treatment and harvested at ZT 6h. Oxygen partial pressure was measured with the NeoFox Sport O2 sensor and probe (Ocean Optics). The dissolved oxygen content in the water covering the plants remained above 18% (v/v) for the 2 h duration of the stress treatment

value: hairy root culture 1cm root tips

value: polyA RNA


title: TOTCONSLSHOOTB [B01_TOTAL]

extract_protocol:
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


title: RNA_Asparagus_10days_leaf_rep2

source_name: mRNA

extract_protocol: RNA-seq: 1.3 ug of total RNA was prepared for sequencing with the Illumina Truseq mRNA Stranded Library Kit, following the manufacturer's protocol


title: ChIP_Brachypodium_7days_leaf_H3K4me1


title: ChIP_Brachypodium_7days_leaf_H3


title: RNA_Brachypodium_7days_leaf_rep2


title: FGR2_mid_2

city: Shanghai

first: Li

last: Ma

email: mary_wyyx@126.com, mary_wyyx@163.com

address: Siping road

country: China

institute: Tongji University

data_processing:
Illumina 1.5
Sequenced reads were mapped to GENECODE vm14 reference genome using hisat2.
Reads count were calculated using gfold(V1.1.4) with default parameters relative to input samples.
Genome_build: GENECODE vm14
Supplementary_files_format_and_content: Cnt files are generated using gfold count function.

organism: Mus musculus

source_name: mid-brain

extract_protocol:
Brain were removed, flash frozen on dry ice, and RNA was harvested using Trizol reagent. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries.
RNA libraries were prepared for sequencing using standard Illumina protocols

value: brain

value: post natal day 3

value: FGR


title: RNA_Asparagus_10days_leaf_rep1


title: TOTCONHAIRYROOTT6A [A17_TOTAL]


title: INRNACONSPSHOOTD [IN_D07]

value: Solanum pennellii LA0716


title: INRNACONSPROOTC [IN_C15]


title: RNA_Brachypodium_7days_leaf_rep1


title: FGR1_mid_1


title: INRNASUBSPROOTC [IN_C16]


title: ChIP_Asparagus_10days_leaf_H3


title: ChIP_Rpo2_input_Sac_log_rep1

organism: Sulfolobus acidocaldarius

extract_protocol:
Crosslinked cells were lysed by sonication, and the extract was clarified by centrifugation. Protein-DNA complexes were purified from the extract with antisera. After reversal of crosslinks, ChIP DNA and input DNA were extracted with phenol:chloroform:isoamyl alcohol and then precipitated in ethanol.
Libraries were constructed using Illumina TruSeq DNA Sample Preparation Kits and were sequenced on the Illumina GAIIx platform.

value: DSM 639

value: Wild type

tag: chip antibody


title: SP-gDNA [SP-gDNA]

extract_protocol:
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

value: Tagmented libraries


title: ChIP_Rpo2_IP_Sac_log_rep1

value: Rpo2 (custom)


title: INRNACONSPSHOOTC [IN_C07]

value: Random primed libraries


title: 150340

type: genomic

city: NICE

first: Thibault

last: FABAS

address: 28 av Valombrose

country: France

institute: CHU Nice

hyb_protocol: DNA was hybridized with the hybridization kit according to the Agilent protocol.

scan_protocol: We used a SureScan provided by Agilent and controled by SureScan Software.

data_processing: The data was processed on Cytogenomics 2.9.1.3 using the Default Analysis Method - CGH v2 proposed by Agilent.

organism: Homo sapiens

source_name: chromophobe rcc

label_protocol: DNA was labeled with the sureTag labeling kit according to the Agilent protocol.

extract_protocol: DNA was extracted from fresh, frozen or formol fixed-paraffin embedded fragments using Qiamp DNA mini kit.

value: Chromophobe renal cell carcinoma

source_name: Female DNA control provided in SureTag Labeling kit

extract_protocol: DNA was provided in SureTag Labeling kit.

tag: sample type

value: Commercial DNA from cell lines


title: ctrl_mid_1

value: wild type


title: TRASUBHAIRYROOTT6A [A18_TRAP]

value: Solanum lycopersicum cv. M82

value: TRAP RNA


title: INRNACONSPROOTB [IN_B15]

value: 7 days-old seedling 1 cm roots tips

value: nuclear RNA


title: ATAC_Populus_leaf_Input

organism: Populus trichocarpa

extract_protocol: ATAC-seq libraries were prepared following the ATAC-seq protocol  as previously described in (Lu et al. 2016).

value: Stettler

value: leaf


title: INRNASUBSPSHOOTC [IN_C08]

value: 7 days-old seedling shoot apical meristem


title: ATACSUBSPSHOOT1 [ATAC-E08]

description: processed data file: ATAC_SP_SH_SUB.bw

extract_protocol:
Nuclei were purified from frozen and pulverized tissue as previously described for A. thaliana (Wang and Deal, 2015) with minor modifications including the use of a 30 µm filter to exclude 30 to 70 µm cellular debris from the crude extract and extend centrifugation times. Tissue was resuspended in an ice-cold mortar containing 10 mL of freshly prepared nuclei purification buffer (NPB: 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, pH, 7.0) containing 200 uL Protease Inhibitor Cocktail (0.4X, Sigma, P9599) per 50 mL of buffer. The homogenized extracts were filtered through a 30 µM nylon mesh to remove cell debris and centrifuged at 1000 x g for 15 min at 4° C to pellet nuclei. Nuclei were resuspended in 1 mL of NPB and 25 µL of M-280 streptavidin-coated Dynabeads (Life Technologies, catalog # 11205D) were added to the nuclei. This mixture was slowly rotated in a cold room at 4° C for 30 min. The nuclei/beads suspension was diluted to 14 mL with NPB supplemented with 0.1% (v/v) Triton X-100 (NPB-T), in a 15 mL Falcon tube, mixed thoroughly and placed in a 15 ml magnet (adapted NEB 50 mL tube magnet) to capture bead-bound nuclei for 1 min at 4° C. The supernatant was carefully removed using a plastic Pasteur pipette, taking care to remove bubbles to avoid disturbing the beads. Beads were resuspended in 14 ml of cold NPB-T, placed on a rotating mixer for 30 sec, and then placed back in the 15 ml magnet to capture the beads-nuclei at 4° C for 1 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube
Tagmentation using Tn5 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


title: INRNACONSPSHOOTB [IN_B07]


title: ATACSUBSPROOT1B [ATAC-D21]

description: processed data file: ATAC_SP_R_SUB.bw


title: TRACONHAIRYROOTT6A [A17_TRAP]

value: hairy root culture 1cm root tips


title: ATAC_Populus_leaf_rep1


title: ATACSUBSPROOT1 [ATAC-D16]

description: processed data file: ATAC_SP_R_SUB.bw

value: Solanum pennellii LA0716


title: ChIP_Populus_leaf_H2A.Z


title: ATACSUBSLSHOOT1 [ATAC-D06]

description: processed data file: ATAC_SL_SH_SUB.bw

treatment_protocol:
Transgenic Solanum seeds from transgenic lines were surface sterilized in 50% (v/v) bleach solution for 5 min (S. pennellii) or 20 min (S. lycopersicum) and then rinsed three times with sterile distilled water. Seedlings were grown on plates (10 cm x 10 cm) containing full-strength Murashige and Skoog standard medium (MS) agar (1% w/v) and 1% w/v sucrose, during 7 days in a growth chamber (16 h day / 8 h night;  at 22°C/22°C day/night; 60-65 μEm-2s-1). For submergence treatment ("SUB"), plates were opened and placed horizontally under 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm) and shoot apical meristems were harvested at ZT 6h (2h before relative noon). Control plates ("CON") were not opened, but positioned similarly horizontally and also harvested at ZT 6h
Four independent biological replicates were grown for each species. For whole plant submergence, plates were placed horizontally, opened and the seedlings covered with 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm; all four species) and the shoot apical meristem region (Solanum species) were harvested at ZT 6h (2h before relative noon). Control plates were not opened, but positioned horizontally for the duration of the treatment and harvested at ZT 6h. Oxygen partial pressure was measured with the NeoFox Sport O2 sensor and probe (Ocean Optics). The dissolved oxygen content in the water covering the plants remained above 18% (v/v) for the 2 h duration of the stress treatment


title: ATACCONSPROOT1 [ATAC-E15]

description: processed data file: ATAC_SP_R_CON.bw


title: ATACCONSPSHOOT1 [ATAC-D07]

description: processed data file: ATAC_SP_SH_CON.bw


title: ChIP_Populus_leaf_Input


title: CBS_Keratin_96h_I

city: Ribeirão Preto

first: Pablo

middle: Rodrigo

last: Sanches

state: SP

address: Av. Bandeirantes, 3900

country: Brazil

institute: University of São Paulo

data_processing:
FastQC v0.11.7 was used to visualize the libraries quality before and after trimming.
Trimmomatic v0.36 was used to remove adapters and quality filtered setting the cutoff threshold for average base quality score at 15 over a window of 4 bases. Reads shorter than 36 bases post-trimming were excluded.
Filtered reads were mapped onto the T. rubrum genome using the STAR v2.5.2b.
Gene level read-counts were quantified using STAR’s –quantMode GeneCounts parameter.
The data were analyzed using the R package DESeq2. The samples were normalized, and the dispersions were estimated using the default settings. The package was then used to identify differentially expressed genes for each pairwise comparison of interest. Genes with an adjusted P value of < 0.05 and a log2(foldchange) >= 1.5 (upregulation) or <= -1.5 (downregulation) were called as differentially expressed.
Genome_build: Broad T. rubrum strain CBS118892 genome
Supplementary_files_format_and_content: tab-delimited text files include reads count values for each sample and DESeq output in CSV format containing mean expression, fold changes and p value.

organism: Trichophyton rubrum

source_name: CBS 118892 - Keratin - 96 hours, replicate I

growth_protocol: For RNA-seq libraries assembly, T. rubrum strain CBS118892 was maintained on malt extract agar [MEA; 2% (w/v) glucose, 2% (w/v) malt extract, 0.1% (w/v) peptone (w/v), pH 5.7] for 17 d at 28 °C.

extract_protocol:
The obtained mycelium was harvested, frozen in liquid nitrogen, and stored at - 80 °C until RNA isolation was performed. Total RNA was isolated using an Illustra RNAspin mini isolation kit (GE Healthcare, Chicago, IL, USA) according to manufacturer’s instructions.
RNA libraries were prepared for sequencing using standard Illumina protocols.

treatment_protocol: Approximately 10-6 conidia mL-1 was inoculated into 100 mL Sabouraud dextrose broth [SDB; 2% (w/v) glucose, 1% (w/v) peptone], and incubated under agitation at 28 °C for 96 h under agitation. Mycelia were aseptically transferred into 100 mL minimal medium (MM) at pH 5.0 containing 70 mM nitrate and either 50 mM glucose (control) or 0.5% (w/v) bovine keratin (treatment) as the carbon source. The cultures were incubated under agitation at 28 °C for 24 h, 48 h, or 96 h.

tag: treatment

value: Keratin - 96 hours


title: CBS_Keratin_96h_II

source_name: CBS 118892 - Keratin - 96 hours, replicate II


title: CBS_Keratin_48h_II

source_name: CBS 118892 - Keratin - 48 hours, replicate II

value: Keratin - 48 hours


title: TOTSUBSPROOTF4 [FF8_TOT]

extract_protocol:
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

value: polyA RNA


title: CBS_Keratin_96h_III

source_name: CBS 118892 - Keratin - 96 hours, replicate III


title: ATACCONSPROOT2 [ATAC-B15]

description: processed data file: ATAC_SP_R_CON.bw

value: Tagmented libraries


title: CBS_Keratin_48h_I

source_name: CBS 118892 - Keratin - 48 hours, replicate I


title: ChIP_Populus_leaf_H3K4me3


title: ATAC_Populus_leaf_rep2


title: ATACCONSPSHOOT2 [ATAC-B07]

description: processed data file: ATAC_SP_SH_CON.bw


title: TOTCONSPROOTF4 [FF7_TOT]

value: Random primed libraries


title: ATACSUBHAIRYROOT2B [ATAC-C22]

description: processed data file: ATAC_SL_H_SUB.bw

treatment_protocol:
Hairy root lines were generated as described (Ron et al. 2014) and grown on horizontal plates (10 cm x 10 cm) containing full-strength MS with vitamins, with agar (1% w/v) and 3% w/v sucrose, 200 mg/L kanamycin and 200 mg/L cefotaxime. Subcloned roots were grown horizontally for 7 days in a growth chamber (16 h day / 8 h night;  at 22°C/22°C day/night; 60-65 μEm-2s-1) prior to the harvest. For submergence treatment ("SUB"), plates were opened and placed horizontally under 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm) were harvested at ZT 6h (2h before relative noon). Control plates ("CON") were not opened, but positioned similarly horizontally and also harvested at ZT 6h
Four independent biological replicates were grown for each species. For whole plant submergence, plates were placed horizontally, opened and the seedlings covered with 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm; all four species) and the shoot apical meristem region (Solanum species) were harvested at ZT 6h (2h before relative noon). Control plates were not opened, but positioned horizontally for the duration of the treatment and harvested at ZT 6h. Oxygen partial pressure was measured with the NeoFox Sport O2 sensor and probe (Ocean Optics). The dissolved oxygen content in the water covering the plants remained above 18% (v/v) for the 2 h duration of the stress treatment


title: ChIP_Populus_leaf_H3K56ac


title: CBS_Keratin_24h_II

source_name: CBS 118892 - Keratin - 24 hours, replicate II

value: Keratin - 24 hours


title: CBS_Keratin_24h_III

source_name: CBS 118892 - Keratin - 24 hours, replicate III


title: RNA_Sac_fadR_rep2

extract_protocol:
RNA was extracted from cells using the method described previously (Samson et al., 2008). After treatment with DNase, the RNA was ethanol-precipitated and dissolved in DEPC-treated water.
Libraries were constructed using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. Sequencing was performed on the Illumina NextSeq 500 platform.

value: DpyrEF DfadR

tag: developmental stage

value: Log-phase cells


title: TRASUBSPROOTF3 [FF6_TRAP]


title: ChIP_Populus_leaf_H3K36me3

extract_protocol: ChIP-seq: ChiP-seq was performed following the methods of Zhang et al (2007) (https://www.ncbi.nlm.nih.gov/pubmed/17439305).  Tissues were cross-linked with 1% formaldehyde and chromatin was sonicated, followed by immunoprecipitation.


title: TRASUBSPROOTF4 [FF8_TRAP]

extract_protocol:
TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (Townsley et al 2014)
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


title: INRNASUBSPROOTD3 [IN_D21_redo]

extract_protocol:
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


title: CBS_Keratin_48h_III

source_name: CBS 118892 - Keratin - 48 hours, replicate III


title: TOTSUBSPROOTF3 [FF6_TOT]


title: ChIP_Populus_leaf_H3

source_name: DNA


title: TRACONSPROOTF4 [FF7_TRAP]


title: ChIP_Populus_leaf_H3K27me3


title: RNA_Sac_fadR_rep1


title: TRACONSPROOTF3 [FF5_TRAP]


title: ChIP_Populus_leaf_H3K4me1


title: CBS_Control_96h_III

source_name: CBS 118892 - Control - 96 hours, replicate III

value: Control - 96 hours


title: CBS_Control_96h_II

source_name: CBS 118892 - Control - 96 hours, replicate II


title: RNA_Populus_leaf_rep2


title: CBS_Control_96h_I

source_name: CBS 118892 - Control - 96 hours, replicate I


title: TOTCONSPROOTF3 [FF5_TOT]

source_name: 7 days-old seedling 1 cm roots tips


title: RNA_Sac_MW001_rep2

source_name: Microorganism

value: DpyrEF


title: ChIP_ClsN_input_Sac_fadR_rep2

extract_protocol:
Crosslinked cells were lysed by sonication, and the extract was clarified by centrifugation. Protein-DNA complexes were purified from the extract with antisera. After reversal of crosslinks, ChIP DNA and input DNA were extracted with phenol:chloroform:isoamyl alcohol and then precipitated in ethanol.
Libraries were constructed using Illumina TruSeq ChIP Library Preparation Kits and were sequenced on the Illumina NextSeq 500 platform.


title: RNA_Populus_leaf_rep1

source_name: mRNA


title: RNA_Sac_MW001_rep1


title: TOTCONSPROOTF2 [FF4_TOT]

value: 7 days-old seedling 1 cm roots tips


title: TRACONSPROOTF2 [FF3_TRAP]

value: TRAP RNA


title: ChIP_ClsN_input_Sac_fadR_rep1


title: TRASUBSPROOTF2 [FF4_TRAP]


title: CBS_Control_48h_II

source_name: CBS 118892 - Control - 48 hours, replicate II

value: Control - 48 hours


title: ATAC_Setaria_7days_leaf_Input

organism: Setaria viridis

value: leaf


title: ATAC_Setaria_7days_leaf_rep2


title: CBS_Control_48h_III

source_name: CBS 118892 - Control - 48 hours, replicate III


title: ChIP_ClsN_input_Sac_MW001_rep2


title: CBS_Keratin_24h_I

source_name: CBS 118892 - Keratin - 24 hours, replicate I


title: CBS_Control_48h_I

source_name: CBS 118892 - Control - 48 hours, replicate I


title: ATAC_Setaria_7days_leaf_rep1


title: ChIP_ClsN_input_Sac_MW001_rep1


title: TRASUBSPROOTF1 [FF2_TRAP]


title: ChIP_ClsN_IP_Sac_fadR_rep2

value: ClsN (custom)


title: ChIP_Setaria_7days_leaf_Input


title: ChIP_Setaria_7days_leaf_H2A.Z


title: CBS_Control_24h_II

source_name: CBS 118892 - Control - 24 hours, replicate II

value: Control - 24 hours


title: ChIP_ClsN_IP_Sac_fadR_rep1


title: CBS_Control_24h_I

source_name: CBS 118892 - Control - 24 hours, replicate I


title: ChIP_Setaria_7days_leaf_H3K4me1


title: ChIP_ClsN_IP_Sac_MW001_rep2


title: ChIP_ClsN_IP_Sac_MW001_rep1


title: TOTCONSPROOTF1 [FF1_TOT]

value: Solanum pennellii LA0716


title: TOTSUBSPROOTF2 [FF3_TOT]


title: ChIP_Setaria_7days_leaf_H3K4me3


title: TRACONSPROOTF1 [FF1_TRAP]


title: TRASUBSPSHOOTE [E04_TRAP]

source_name: 7 days-old seedling shoot apical meristem


title: ChIP_Setaria_7days_leaf_H3K56ac


title: RS-MT-SUB4 [MT-RSSUBHAIRYROOTMT4]

description: processed data file: MT-RS-SUB.merged.bw

organism: Medicago truncatula

source_name: 4 week-old composite plant 1 cm root tips

extract_protocol:
TRAP was performed as previously described (Mustroph et al 2009, Reynoso et al 2015) with the following modifications: ɑ-FLAG conjugated IgG Dynabeads were used for binding; after magnetic collection and washing the polysomes were removed from the magnetic beads by addition of Lysis and Binding Buffer (LBB) buffer for polyA mRNA isolation using biotinylated oligo-dT primers and streptavidin magnetic beads (NEB) (Townsley et al 2014)
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

treatment_protocol:
M. truncatula seeds were surface-sterilized by incubating in concentrated sulfuric acid for 8 minutes with gentle stirring, washing 3 times with 4°C sterile, distilled water, then 4-8 minutes in 3% (w/v) hypochlorite (diluted bleach), washing 4 times with sterile, distilled water, and finally placing the seeds onto moist filter paper. Seeds were germinated without vernalization on moist filter papers in inverted Petri dishes wrapped with surgical tape wrapping. These were kept in the growth room in the dark at 20°C for 2 days. Hairy root composite M. truncatula plants were initiated by injecting with A. rhizogenes K599. To do so, the day before injection, a volume of fresh overnight culture of A. rhizogenes K599 carrying the 35S:His6-FLAG-GFP-MtRPL18-3 plasmid was used to inoculate  5 ml of Yeast Extract Beef media (5 g/L tryptone, 1 g/L yeast extract, 5 g/L nutrient broth, 5 g/L sucrose, 0.49 g/L MgSO4x7H2O, 15 g/L agar, pH=7.2) additionally containing 100 mg/L Spectinomycin and grown overnight at 28°C at 200 rpm. When the OD600 was equal to 1.0, cultures were centrifuged at 5000 rpm for 5 min. The supernatant was poured off and the pellet was resuspended in Injection Media (IM) (1X PBS, 100 uM acetosyringone, 1/10000 (v/v) Silwet). Seedlings germinating for 2.5-days were injected by placing them in 5 mL of IM-resuspended A. rhizogenes poured out on a Petri dish and stabbing the root a few times with an 18G1 needle. Additionally, 1-2 mm of the primary root tip was cut off. Injected seedlings were moved to slanted Fahräeus Media (FM) plates (0.5 mM MgSO4, 0.7 mM KH2PO4, 0.8 mM Na2HPO4, 50 nM FeEDTA, 0.5 mM NH4NO3, 1mM CaCl2, 0.1 mg of MnSO4, CuSO4, Zn SO4, H3BO3, and Na2MoO4, 8 g/L Phytoblend agar, pH 6.5) with no selection and were grown horizontally for 3 days, and then were moved to FM plates with 5 mg/L phosphinothricin and were grown vertically for 3 wk before transfer to 1X MS media without vitamins (1% w/v agar, 1% w/v sucrose). Plants were grown vertically for 1 wk before being used for the submergence experiment. The day before the experiment was performed, root tips from one plate were collected and visualized on a fluorescence stereomicroscope to check for GFP expression. Transformation efficiency was calculated as the percentage of root tips with ubiquitous GFP expression
Four independent biological replicates were grown for each species. For whole plant submergence, plates were placed horizontally, opened and the seedlings covered with 5 cm of autoclaved distilled water at ZT 4h. Root tips (apical 1 cm; all four species) and the shoot apical meristem region (Solanum species) were harvested at ZT 6h (2h before relative noon). Control plates were not opened, but positioned horizontally for the duration of the treatment and harvested at ZT 6h. Oxygen partial pressure was measured with the NeoFox Sport O2 sensor and probe (Ocean Optics). The dissolved oxygen content in the water covering the plants remained above 18% (v/v) for the 2 h duration of the stress treatment

value: Medicago truncatula Jemalong A17

value: 35S:His6:FLAG-GFP-MtRPL18-3 injection transformed root tips

value: Ribosome footprints

value: Riboseq RNA


title: RS-MT-CON4 [MT-RSCONHAIRYROOTMT4]

description: processed data file: MT-RS-CON.merged.bw

value: 4 week-old composite plant 1 cm root tips


title: TRACONSPSHOOTE [E03_TRAP]

value: 7 days-old seedling shoot apical meristem

value: Random primed libraries


title: HiC_Sac_fadR_rep2

extract_protocol:
Cells were crosslinked, permeabilized, and treated with HindIII to digest cellular DNA. The DNA was end-labeled with biotin and subjected to priximity ligation. After reversal of crosslinks, the DNA was extracted with phenol:chloroform:isoamyl alcohol, precipitated in isopropanol, and sonicated.
Libraries were constructed using NEBNext Ultra DNA Library Prep Kit for Illumina. Biotin-labeled DNA was purified with streptavidin before PCR. Sequencing was performed on the Illumina NextSeq 500 platform.

value: DpyrEF DfadR

value: Log-phase cells


title: HiC_Sac_fadR_rep1


title: designed_library_25-100_length_utrs

city: Seattle

first: Paul

last: Sample

state: Washington

address: West Stevens Way NE

department: Electrical Engineering

institute: University of Washington

description: reporter RNA

data_processing:
cutadapt --length 59 --minimum-length 59 -o r1.fastq -p r2.fastq
Combine r1.fastq sequences (UTRs) and r2.fastq (UMIs) into a single CSV for Bartender
bartender_single_com -f input_csv -o output_file.csv  -c 2 -d 2 -z -1
Calculate relative reads within fractions and then, using these values, calculate relative reads per fraction for a given UTR. Calculate mean ribosome load (MRL) by multiplying each fraction’s relative reads by the number of ribosomes associated with each fraction and then sum the values.
Supplementary_files_format_and_content: CSV files contain 5' UTR sequences, read counts per fraction, relative counts, and MRL ('rl').

source_name: eGFP Variable Length 5' UTRs

growth_protocol: HEK293T cells were cultured on ECM-coated plates in Dubelco's DMEM media supplemented with FBS (5%), penicillin (.05%), and streptomycin (.05%)

extract_protocol:
Salt solution (10x): 100 mM NaCl, 100 mM MgCl2,100 mM Tris-HCl pH 7.5, and RNase-free water (Zuccotti P, Modelska A 2016). Wash buffer: 100 µg/ml cycloheximide in RNase-free DPBS (10 ml per plate). Lysis buffer: 1x salt solution, 1% of 20% Triton X-100, 1 mM DTT, 0.2 U/µl SUPERase-In (Thermo Fisher Scientific), 100 µg/ml cycloheximide. Wash buffer and lysis buffer were chilled throughout the protocol. After 12 hours of growth at 37 °C, cells were placed on ice and media was aspirated. Translating ribosomes were halted by adding 5 ml of wash buffer and were then placed at 37 °C for 5 minutes followed by aspiration on ice. Washed by adding 5 ml wash buffer and aspirating thoroughly. Added 300 µl of ice-cold lysis buffer, scraped cells, broke up cell clumps by pipetting ~5 times, and then placed suspended cells into a pre-chilled microcentrifuge tube. Let sit for 10 minutes on ice and then triturated by passing through a 25-G needle 10 times [1]. Spun at 16,000 x g for 5 minutes to pellet cell debris and nuclei. Saved supernatant and added 1.5 µl of 1 U/µl DNase I (final concentration of 0.005 U/µl) and placed on ice for 30 minutes. Either stored at -80 °C or proceeded directly to polysome profiling.
For both the eGFP and mCherry library, randomized 50-mer oligos were Gibson assembled to serve as 5' UTRs for each CDS. To create the designed library, oligos were made via oligo synthesis array (CustomArray Inc.) and cloned into the eGFP vector.For all libraries, 25 nucleotides of defined sequence preceed the inserted 5' UTRs.

treatment_protocol: HEK293T cells were plated on 10cm cell culture dishes 24 hours before transfection (1 - 2 million per plate). At 60% to 80% confluency, cells were transfected with 14.5 µg of library mRNA using Lipofectamine MessengerMAX (Thermo Fisher Scientific) following the manufacturer’s protocol. Washed plates with 10 ml 1x DBPS and 10 ml media (DMEM with 10% FBS and 1% Penicillin-Streptomycin) after one hour of incubation. Cells were lysed 12 hours after transfection.

tag: cell type


title: TOTSUBSPSHOOTE [E04_TOTAL]

value: polyA RNA


title: ChIP_Setaria_7days_leaf_H3K27me3


title: CBS_Control_24h_III

source_name: CBS 118892 - Control - 24 hours, replicate III


title: RS-MT-SUB3 [MT-RSSUBHAIRYROOTMT3]


title: HiC_Sac_MW001_rep2

value: DpyrEF


title: TOTCONSPSHOOTE [E03_TOTAL]


title: ChIP_Setaria_7days_leaf_H3K36me3

source_name: DNA


title: RS-MT-CON3 [MT-RSCONHAIRYROOTMT3]


title: TOTSUBSPSHOOTD [D04_TOTAL]


title: TOTSUBSPROOTF1 [FF2_TOT]

value: 7 days-old seedling 1 cm roots tips


title: RNA_Setaria_7days_leaf_rep2

extract_protocol: RNA-seq: 1.3 ug of total RNA was prepared for sequencing with the Illumina Truseq mRNA Stranded Library Kit, following the manufacturer's protocol


title: HiC_Sac_MW001_rep1


title: RNA_Sac_sta_rep1

value: DSM 639

value: Wild type

value: Stationary-phase cells


title: RNA_Setaria_7days_leaf_rep1

value: leaf


title: RS-MT-CON1 [MT-RSCONHAIRYROOTMT1]


title: ATAC_Barley_7days_leaf_Input

organism: Hordeum vulgare

value: Morex


title: RNA_Sac_sta_rep2


title: RNA_Sac_log_rep2


title: TRACONSPSHOOTD [D03_TRAP]

value: Solanum pennellii LA0716

value: TRAP RNA