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| Status |
Public on May 07, 2023 |
| Title |
HSFA3-ACC+56h-R2 |
| Sample type |
SRA |
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| Source name |
seedlings
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| Organism |
Arabidopsis thaliana |
| Characteristics |
developmental stage: 6 day old seedling
treatment: ACC+56h
genotype: pHSFA3::3xFlag-HSFA3
chip antibody: DYKDDDDK isolation kit (Miltenyi Biotec)
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| Treatment protocol |
For HS treatments for ChIP-seq, 4 d-old seedlings were exposed to a two-step acclimation (ACC) protocol consisting of 37°C for 1 h, 23°C for 90 min and 44°C for 45 min. Samples for ChIP-seq were harvested 4 h, 28 h, or 52 h after the end of the ACC treatment. Non-heat stressed (NHS) samples were harvested at the same time as the ACC+4h samples. For maintenance of acquired thermotolerance assay, 4 d-old seedlings were exposed to the same ACC protocol as described above and 3 d later, to a triggering heat stress of 44°C of 70-110 min as indicated. Seedlings were imaged 14 d after end of ACC.
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| Growth protocol |
Seedlings were grown on GM medium (1 % [w/v] glucose) under a 16 h/8 h light/dark cycle at 23/21°C. pHSFA3::3xFlag-HSFA3 hsfa3 was described previously (Friedrich et al., 2021; https://doi.org/10.1038/s41467-021-23786-6).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linking and immunoprecipitation of samples was performed as described previously (Kaufmann et al., 2010; Friedrich et al., 2021). Briefly, 3xFlag-HSFA2 and 3xFlag-HSFA3 seedlings were harvested at the indicated time points (3 replicates) and immediately cross-linked under vacuum in 25 ml ice-cold MC buffer (1 % (v/v) formaldehyde) for 2 x 10 min. For chromatin extraction, frozen tissue was ground, resuspended in 25 ml M1 buffer, and filtered through Miracloth mesh (Merck), washed five times in 5 ml M2 buffer, and washed once in 5 ml M3 buffer with a 10 min, 4°C, 1,000 g centrifugation step in between each washing step. The chromatin pellet was resuspended in 1 ml Sonication buffer and sonified using a Diagenode Bioruptor (17 cycles of 30 sec on/off at low intensity settings). For 3xFlag-HSFA2/A3 ChIP, chromatin was incubated with anti-DYKDDDDK paramagnetic beads (Miltenyi Biotec) for 1.5 h at 4°C and recovered with DYKDDDDK isolation kit (Miltenyi Biotec).
DNA libraries for each sample were prepared from isolated chromatin using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB) in combination with NEBNext Multiplex Oligos for Illumina (NEB) according to the manufacturer’s instructions. No size selection was performed after adaptor ligation. DNA was amplified by PCR with 11 amplification cycles. Library concentration and fragment size distribution were checked using D1000 ScreenTape with TapeStation bioanalyzer (Agilent). Library quantification was done with the NEBNext Library Quant Kit for Illumina (NEB) according to the manufacturer’s instructions. Pooled libraries were sequenced on a NextSeq 500 (Illumina), 75 bp SE reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
HSFA3-ACC+56h
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| Data processing |
Reads were mapped against the Arabidopsis thaliana reference genome (TAIR10) using bwa mem (Li, 2013; http://arxiv.org/abs/1303.3997) version 0.7.12-r1044.
Mappings were sorted and indexed using samtools (Li et al., 2009; http://doi.org/10.1093/bioinformatics/btp352) version 1.3.1.
RPGC normalized coverage tracks were generated using the deepTools (Ramírez et al., 2016; http://doi.org/10.1093/nar/gkw257) version 1.50 bamCoverage function.
Genome_build: Arabidopsis thaliana TAIR10
Supplementary_files_format_and_content: Normalized coverage files in bigWig format.
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| Submission date |
Dec 21, 2021 |
| Last update date |
May 07, 2023 |
| Contact name |
Isabel Bäurle |
| E-mail(s) |
isabel.baeurle@uni-potsdam.de
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| Phone |
+49 331 9772647
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| Organization name |
Universität Potsdam
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| Department |
Institut für Biochemie und Biologie
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| Street address |
Karl-Liebknecht-Str. 24-25
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| City |
Potsdam |
| ZIP/Postal code |
14476 |
| Country |
Germany |
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| Platform ID |
GPL19580 |
| Series (2) |
| GSE192427 |
HSFA2 and HSFA3 binding after heat acclimation [ChIP-seq] |
| GSE192431 |
HSFA2 and HSFA3 binding after heat acclimation |
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| Relations |
| BioSample |
SAMN24289011 |
| SRA |
SRX13476170 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5746765_2-A3-56h_S8.SeqDepthNorm.bw |
45.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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