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| Status |
Public on Sep 19, 2019 |
| Title |
TRASUBSPSHOOTC [C04_TRAP] |
| Sample type |
SRA |
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| Source name |
7 days-old seedling shoot apical meristem
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| Organism |
Solanum pennellii |
| Characteristics |
cultivar: Solanum pennellii LA0716
line: 35S:His6:FLAG-GFP-AtRPL18-1
tissue: 7 days-old seedling shoot apical meristem
experiment type: Random primed libraries
molecule subtype: TRAP RNA
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| 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
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| 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
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| Extracted molecule |
total RNA |
| Extraction 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
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| 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
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| Submission date |
Mar 21, 2019 |
| Last update date |
Sep 21, 2019 |
| Contact name |
Roger B Deal |
| E-mail(s) |
roger.deal@emory.edu
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| Phone |
404-727-8087
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| Organization name |
Emory University
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| Department |
Biology
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| Street address |
1510 Clifton Rd NE
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
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| Platform ID |
GPL26332 |
| Series (1) |
| GSE128680 |
Multiscale gene expression profiling of 1 cm root tips of control and submerged Oryza sativa, Medicago truncatula, Solanum lycopersicum and Solanum pennellii seedlings |
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| Relations |
| BioSample |
SAMN11191879 |
| SRA |
SRX5554207 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3682238_C04-TRAP.bw |
5.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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