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Status |
Public on Aug 21, 2024 |
Title |
IZE explants, E0 10d a |
Sample type |
SRA |
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Source name |
immature zygotic embryos
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Organism |
Arabidopsis thaliana |
Characteristics |
tissue: immature zygotic embryos time: 10 day developmental stage: non-embryogenic culture genotype: Columbia-0 treatment: lack media: E0
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Treatment protocol |
IZE explants were treated by 5 or 10 day with 5.0 μM of 2,4-D (Sigma-Aldrich #D7299) (EA medium) or with TSA (ET) (Sigma Aldrich; #T1952) at a concentration of 1.0 μM.
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Growth protocol |
Immature zygotic embryos (IZEs) at the cotyledonary stage of development were used as explants for the in vitro culture. A basal E0 medium contained 3.2 g L-1 of B5 micro and macro-elements (Duchefa Biochemie; #G0210), 20 g L-1 sucrose and 8 g L-l agar, pH 5.8. A standard medium for SE induction (EA) contained 5.0 μM of 2,4-D (Sigma-Aldrich #D7299). In addition, E0 medium supplemented with TSA (ET) (Sigma Aldrich; #T1952) at a concentration of 1.0 μM was applied for SE induction. Plant materials that were grown in sterile conditions were kept at 23°C under a 16 h photoperiod of 40 µM m-2 s-1 white, fluorescent light
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Extracted molecule |
total RNA |
Extraction protocol |
An RNAqueous® Total RNA Isolation Kit (ThermoScientific) was used to isolate total RNA from the IZE explants induced on different media (E0, EA, ET) for 5 and 10 days. The freshly isolated tissues were wiped in frozen mortars. Depending on the age of the culture, from 250 (5th day) to 100 (10th day) explants were used for RNA isolation per repetition. RNA isolation, library preparation, and sequencing were produced in three biological replicates. The concentration and purity of RNA samples were evaluated with an ND-1000 spectrophotometer (NanoDrop Technologies). The integrity of the RNA was determined using an Agilent 2100 Bioanalyzer and Agilent RNA 6000 Nano chips (Agilent Technologies, Santa Clara, USA). RNA samples were treated with RNase-Free DNase and then purified using the Acid-Phenol: Chloroform with ammonium acetate method (ThermoScientific). The sequencing libraries were prepared using Illumina ScriptSeq™ Complete Kit (Plant; Illumina, San Diego, USA) following the manufacturer’s protocol. Two micrograms of total RNA per sample were used as an input. Briefly, library prep involved the subsequent steps: ribosomal RNA removal with Ribo-Zero rRNA Removal Reagents (Plant Leaf; Illumina, San Diego, USA) followed by ethanol precipitation of the rRNA-depleted sample, RNA fragmentation, cDNA synthesis, RNA removal, terminal tagging of cDNA followed by bead cleanup, PCR amplification using Illumina indexes and final bead purification. The quality of the prepared Illumina libraries was analyzed using Agilent Bioanalyzer with the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, USA), and the quantities were estimated using a Qubit Fluorometer (Thermo Fisher Scientific, Waltham, USA). For cluster generation, the libraries were pooled with equimolar concentration and sequenced using the Illumina HiSeq 4000 system (Illumina, San Diego, USA) in 2x 76 cycles paired-end (PE) mode with 6 barcoded samples per lane.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 4000 |
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Description |
p_E0_10_a
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Data processing |
Sequencing data were processed to obtain fastq files with bcl2fastq2 Conversion Software v2.19.0 (Illumina, San Diego, USA) including demultiplexing and adapter trimming steps. The quality of the obtained sequencing data was assessed before the analysis and after each of its stages, using the FastQC tool (The Babraham Institue, Cambridge, United Kingdom) and MultiQC (Ewels et al., 2016) tools. All reads were trimmed to the length of 75 bp with BBduk (DOE Joint Genomes Institute, Walnut Creek, Ca, USA). Due to the high quality of the reads, they were only soft filtered and trimmed using Sickle tool (https://github.com/najoshi/sickle). The remaining ribosomal RNA reads were then removed using the SortMeRNA software. Clean reads were mapped to the reference genome sequence assembly GCA_000001735.1 (TAIR10) with the splice-aware aligner STAR (Dobin et al., 2012) using two pass mode and parameters adjusted to the Arabidopsis thaliana genome annotation regarding mates gaps and intron lengths. The quality of mapping was assessed with QualiMap (Okonechnikov et al., 2016) as well as SAMStat (Lassmann et al., 2011). Reads mapping to genes were counted using quantMode in STAR. The analysis of differences in gene expression levels between samples was performed with the DESeq2 tool (Love et al., 2014) assuming negative binomial distribution and applying general linearized model with beta prior shrinkage. Assembly: GCA_000001735.1 (TAIR10) Supplementary files format and content: Raw feature counts with relation to genes: gene symbols are in rows, samples in columns.
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Submission date |
Feb 06, 2024 |
Last update date |
Aug 21, 2024 |
Contact name |
Barbara Karolina Wójcikowska |
E-mail(s) |
barbara.wojcikowska@us.edu.pl
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Phone |
606245490
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Organization name |
University of Silesia
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Department |
Faculty of Natural Sciences; Institute of Biology, Biotechnology and Environmental Protection
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Street address |
Jagiellonska
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City |
Katowice |
State/province |
Silesia |
ZIP/Postal code |
40-032 |
Country |
Poland |
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Platform ID |
GPL21785 |
Series (1) |
GSE255229 |
Transcriptomic profiling reveals histone acetylation-regulated genes involved in somatic embryogenesis in Arabidopsis thaliana |
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Relations |
BioSample |
SAMN39849218 |
SRA |
SRX23556441 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
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