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| Status |
Public on Jun 17, 2024 |
| Title |
at-endosperm-H3K27m3_replicate1_ler_x_col_Cm |
| Sample type |
SRA |
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| Source name |
endosperm
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| Organism |
Arabidopsis thaliana |
| Characteristics |
tissue: endosperm
cell line: AT F1-Endosperm Landsberg x Columbia TEmob
genotype: Ler (maternal) x TEmob Col (paternal)
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| Treatment protocol |
Material was generated by manually crossing of pistillata (Ler) mutants with pollen of INT line (Col) and the INT line pollinated with Ler
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| Growth protocol |
Seeds were surface sterilized with 70% ethanol and washed three times with sterile water. Sterilized seeds were sown on 1⁄2MS plates containing 0.5% sucrose and 0.8% agar, stratified for 2 days at 4°C and germinated under long-day conditions (16h light/8h darkness) at 21°C. Seedlings were transferred to soil after 10–12 days and grown in phytotrons under long day conditions (day 21°C, night 20°C 70% humidity, 150μE light intensity).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
We coupled the previously published INTACT protocol for endosperm-nuclei purification (Moreno-Romero et al., 2017) with Cleavage Under Targets & Tagmentation (CUT&Tag) (Kaya-Okur et al., 2019). Tissue homogenization and nuclei purification were performed as previously described (Del Toro-De León & Köhler, 2019) except that MgCl2 was replaced by spermidine 0.5 mM in the Honda buffer to prevent pAG-Tn5 activation by residual MgCl2. All steps were performed with streptavidin-bound nuclei. INTACT-purified endosperm nuclei from 250 mg of siliques at 4DAP were resuspended in Antibody150 buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin, 2 mM EDTA pH 7.5). Primary antibody incubation was performed overnight at 4°C with gentle shaking using anti-H3K9me2 (Diagenode Cat. No. C15410060), Anti-Histone H3 (Sigma-Aldrich Cat. No. H9289) or anti-H3K27me3 (Cell Signaling Technology Cat. No. 9733T) antibodies. Secondary antibody incubation was performed with Guinea Pig anti-Rabbit IgG, (Antibodies-Online Cat. No. ABIN101961) for 30 min at RT with Digitonin150 Buffer (20 mM HEPES, pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin). Beads were washed twice with Digitonin150 Buffer and incubated with pAG-Tn5 (EpiCypher Cat No. 15-1017) for 1hr in Digitonin300 Buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin). Beads were washed twice with Digitonin300 Buffer and tagmentation was performed at 37ºC for 1hr with Tagmentation Buffer (20 mM HEPES, pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, 1x Roche cOmplete™ Mini EDTA-free Protease Inhibitor, 0.01% Digitonin, 10 mM MgCl2). Beads were washed with TAPS Buffer (10 mM TAPS, pH 8.5, 0.2 mM EDTA) and the tagmentation reaction was quenched with SDS Release Buffer (10 mM TAPS, pH 8.5, 0.1% SDS). Tagmented chromatin fragments were released by incubation for 1hr at 58ºC. SDS Quench Buffer (0.67% Triton-X 100) was added to the samples and library amplification was performed. We prepared unique index Nextera-compatible libraries with custom-designed index primers. PCR was performed with Non-hot Start Phusion™ High-Fidelity DNA Polymerase (Thermo Scientific™ Cat No. F530S) for 14 cycles. Final DNA library fragment purification was performed with SPRI beads (ratio of 1.3 μl of SPRI beads to 1 μl of PCR product) and washed twice with 80% ethanol. The final library was resuspended in 0.1x TE buffer.
CUT&Tag customized Nextera library
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 1000 |
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| Description |
Arabidopsis thaliana F1-endosperm of Landsberg-erecta pi-1 (female) crossed with TEmob Columbia-0 INT (male)
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| Data processing |
cutNtag: PE reads were trimmed using the trim_galore tool and mapped to the Arabidopsis (TAIR10) genome using Bowtie2 (Langmead & Salzberg, 2012) (parameters --no-unal --no-mixed --no-discordant --phred33 --local --very-sensitive-local). Mapped reads were sorted and indexed using SAMtools (Danecek et al., 2021). Read coverage was estimated and normalized to 1x sequencing depth (reads per genome coverage, RPGC) using the bamCoverage from deepTools (Ramírez et al., 2014) (parameters -bs50 --effectiveGenomeSize 119481543 --normalizeUsing RPGC --skipNAs). Signal-to-noise ratio was normalized with H3 data by calculating the log2 ratio in 50 bins across the genome using bigwigCompare from deepTools. Parent-of-origin methylation was performed as previously described (Moreno-Romero et al., 2016).
Assembly: Arabidopsis thaliana TAIR10
Supplementary files format and content: cutNtag:bigWig parental-specific (for parents LER and COL) coverage files normalised as reads per genomic content (RPGC)
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| Submission date |
Mar 04, 2024 |
| Last update date |
Jun 17, 2024 |
| Contact name |
Juan C Santos-González |
| E-mail(s) |
juan.santos@slu.se
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| Organization name |
SLU
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| Department |
Department of Plant Biology and Forest Genetics
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| Lab |
Köhler's lab
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| Street address |
Almas Allé 5
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| City |
Uppsala |
| State/province |
Uppsala |
| ZIP/Postal code |
75007 |
| Country |
Sweden |
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| Platform ID |
GPL34120 |
| Series (2) |
| GSE260818 |
Epigenetic and transcriptional consequences of chemically induced transposon mobilization in the endosperm (CUT&Tag) |
| GSE260822 |
Epigenetic and transcriptional consequences in the endosperm of chemically induced transposon mobilization in Arabidopsis |
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| Relations |
| BioSample |
SAMN40253821 |
| SRA |
SRX23829850 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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