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Sample GSM4494376

Query DataSets for GSM4494376

Status

Public on Mar 24, 2021

Title

ACTTRAPH

Sample type

SRA

Source name

3-5 days-old seedling 1 cm roots tips

Organism

Solanum lycopersicum

Characteristics

cultivar: M82
line: SlACT2pro:His6:FLAG-GFP-AtRPL18-2
age: 3-5 days
tissue: Roots tips

Growth protocol

growth protocol 1: Transgenic Solanum lycopersicum seeds from transgenic lines were surface sterilized in 50% (v/v) bleach solution for 20 min (S. lycopersicum) and then rinsed three times with sterile distilled water. Seven seeds were plated on 10 cm x 10 cm plates containing full-strength Murashige and Skoog standard medium (MS) agar (1% w/v) and 1% w/v sucrose and germination was scored daily after plating. Seedlings were grown for 3-5 days after germination in a growth chamber (16 h day / 8 h night; at 22°C/22°C day/night; 60-65 ?Em-2s-1) and 1cm primary root tips were harvested and flash frozen into liquid nitrogen.
growth protocol 2: 1-month-old control plants: Transgenic Solanum lycopersicum seeds were germinated on 1xMS media with 1% sucrose and 200ug/ml Kanamycin. After 7d, seedlings were transplanted into pots with Turface Athletic Profile Field & Fairway clay substrate (Turface Athletics, 2017) that was pre-wetted with a nutrient water solution containing 4% nitrogen, 18% phosphoric acid, and 38% soluble potash. Plants were grown in a randomized design for 31 d in a Conviron Growth Chamber at 22°C, 70% RH, 16/8 h light/dark cycle, light intensity of 150-200 µmol/m2/s.
growth protocol 3: Field: Transgenic Solanum lycopersicum seeds were germinated on 1xMS plate (0-7d), screened for correct GFP pattern from root tip, transplanted on soil and grown in a growth chamber for one week (7-14d) , grown in a screenhouse for two weeks (14-28d), and finally transplanted into the field and grown for one month (29-60d) with drip watering and weekly removal of flower buds.
growth protocol 4: Rice seeds from transgenic lines were dehulled and surface sterilized in 50% (v/v) bleach solution for 30 min and then rinsed with sterile distilled water. Seedlings were grown on plates (10 cm x 10 cm) containing half-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 28°C/25°C day/night; 110 ?Em-2s-1). The whole root system was placed immediately into liquid nitrogen upon harvesting.

Extracted molecule

total RNA

Extraction protocol

extract protocol 1: Translating Ribosome Affinity Purification (TRAP) was performed as previously described (Reynoso et al, 2019).
extract protocol 2: Nuclei were purified from frozen and pulverized tissue as previously described (Reynoso et al, 2019). Tissue was resuspended in an ice-cold mortar containing 10 mL of freshly prepared nuclei purification buffer (NPB: 20 mM MOPS, 40 mM NaCl, 90 mM KCl, 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.2 mM spermine, pH, 7.0) containing 200 uL Protease Inhibitor Cocktail (0.4X, Sigma, P9599) per 50 mL of buffer. The homogenized extracts were filtered through a 30 µM nylon mesh to remove cell debris and centrifuged at 1000 x g for 15 min at 4° C to pellet nuclei. Nuclei were resuspended in 1 mL of NPB and 25 µL of streptavidin-coated beads (NEB) were added to the nuclei. This mixture was slowly rotated in a cold room at 4° C for 30 min. The nuclei/beads suspension was diluted to 14 mL with NPB supplemented with 0.1% (v/v) Triton X-100 (NPB-T), in a 15 mL Falcon tube, mixed thoroughly and placed in a 15 ml magnet to capture bead-bound nuclei for 1 min at 4° C. The supernatant was carefully removed using a plastic Pasteur pipette, taking care to remove bubbles to avoid disturbing the beads. Beads were resuspended in 14 ml of cold NPB-T, placed on a rotating mixer for 30 sec, and then placed back in the 15 ml magnet to capture the beads-nuclei at 4° C for 1 min. This wash step was repeated and bead-bound nuclei were resuspended in 1 mL of NPB-T and transferred to a new tube
extract protocol 3: For Rice: TRAP was performed as previously described (Reynoso et al, 2019)
library construction protocol 1: RNA-seq libraries were prepared from poly(A)+ selected total and TRAP mRNA as described by Townsley et al. (2014) for random primer primed libraries.
library construction protocol 2: RNA-seq libraries were prepared from poly(A)+ selected total and TRAP mRNA as described by Townsley et al. (2014) for random primer primed libraries.

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

Solanum lycopersicum cv. M82
Random primed libraries
TRAP RNA
H09
processed data file: Tomato_ATLAS_raw_counts.csv
protocols used: growth protocol 1, extract protocol 1 and library construction protocol 1

Data processing

Library strategy: TRAP-seq
Sequences were pooled, and then trimmed and filtered using Trim Galore! (v0.4.5) with parameter -a GATCGGAAGAGCACA. Trimmed reads were pseudo-aligned to ITAG3.2 transcriptome (cDNA) using Kallisto (v0.43.1), with the parameters -b 100 --single -l 200 -s 30, to obtain count estimates and transcript per million (TPM) values.
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.
Rice TRAP-seq. Barcoded libraries were pooled together and sequenced on Illumina HiSeq 3000 at the UC Davis DNA Technologies Core to obtain 50-bp reads.
Rice TRAP-seq. R/Bioconductor (version 3.2.1) and FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) were used in short read quality control and grep for filtering of oligo dT for PolyA RNA libraries.
Rice TRAP-seq. Sequences were pooled, and then trimmed and filtered using Trim Galore! (v0.4.5) with parameter -a GATCGGAAGAGCACA. Trimmed reads were pseudo-aligned to IRGSP-1.0 transcriptome (CDS, https://rapdb.dna.affrc.go.jp/index.html) using Kallisto (v0.43.1), with the parameters -b 100 --single -l 200 -s 30, to obtain count estimates and transcript per million (TPM) values. Splice variants were summed to assess transcript values.
Genome_build: Tomato: SOLANUM LYCOPERSICUM: ITAG3.20 (ftp://ftp.solgenomics.net/tomato_genome/assembly/build_3.00/)
Genome_build: Rice: IRGSP-1.0 (https://rapdb.dna.affrc.go.jp/download/irgsp1.html)
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.
Supplementary_files_format_and_content: .csv files contain raw counts.

Submission date

Apr 23, 2020

Last update date

Mar 24, 2021

Contact name

Siobhan Brady

E-mail(s)

sbrady@ucdavis.edu

Phone

5307525183

Organization name

University of California Davis