NGS, confocal microscopy and flow cytometry data of CRISPR/Cas9-edited ARE1 and KAT2 genes in transgenic maize in vitro tissues exposed to chromatin modifiers
Description
This dataset contains raw NGS reads (NGS_data folder), fluorescence microscopy photos (Fig7B Microscopy Originals.lif) and flow cytometry data (@Fig6 FACS original data.zip) derived from transgenic maize (Zea mays L.) in vitro tissues exposed to chromatin modifiers. They are presented in research article ˝Variable Alterations in Frequencies of CRISPR/Cas9-Induced Nucleotide Deletion and Insertion in Transgenic Maize Callus Tissues Exposed to Chromatin Modifiers˝. Stable transgenic maize lines harboring active CRISPR/Cas9 vector systems targeting either the abnormal cytokinin response 1 (ARE1) gene or the potassium channel gene (KAT2) were used. Mutagenic events following exposure to elevated temperatures (37 °C for 48 h) and various chromatin-modifying compounds were quantified. As a novel approach, the mutagenic potential of combining chromatin-modifying agents with heat exposure was examined.
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Prior to sequencing, the PCR products from three independent biological replicates per treatment were pooled. Sequencing was performed using an Illumina platform. Raw sequenced reads, >0.2 M per sample, were demultiplexed and adapter-trimmed using NovaSeq Control Software, while FastQ Toolkit (Illumina) was applied to bin reads having a mean quality score of less than 30 and shorter than 50 bp. The BWA software package was applied to map the filtered sequences to the reference sequences. The raw sequence data was analyzed using Integrative Genomics Viewer (IGV) software. Images of DAPI stained cells were acquired using Leica DMI8–Stellaris 5 laser scanning microscope (Leica Microsystems Heidelberg, Germany) using DAPI detection settings. To evaluate DNA content, fixed nuclei suspended in phosphate buffered saline (PBS, pH 7.4) were stained with DAPI (4′,6-diamidino-2-phenylindole (DAPI, AppliChem, Biochemica, Darmstadt, Germany) at a final concentration of 1 μg/mL and incubated on ice for 3 min in the dark. The samples were analyzed using a BD Aria Fusion FACS (BD Biosciences, CA, United States of America), and 10,000 singlet events were acquired per sample using the BD FACSDiva software.
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Funders
- National Research, Development and Innovation OfficeBudapest, BudapestGrant ID: RRF 2.3.1–21-2022- 00007 grant for the ‘National Laboratory Program of Agrobiotechnology and Precision Plant Breeding to Support Food Safety’
- EU’s Horizon 2020 Research and Innovation ProgramGrant ID: 739593