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| Status |
Public on Mar 17, 2020 |
| Title |
Input_A_rep2 |
| Sample type |
SRA |
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| Source name |
Sperm nuclei
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Col-0
genotype: wild type
chip-antibody: none
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sperm nuclei from an ProHTR10:HTR10-Clover line were isolated by FACS and stored at -80°C in Cryopreservation Buffer (50 mM Tris-Cl pH 8.0, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, 1 mM PMSF, 1x Roche protease inhibitor cocktail). Sperm mononucleosomes were generated by micrococcal nuclease (MNase, New England Biolabs) digestion. Aliquots of frozen sperm nuclei were thawed on ice and subsequently subjected to MNase digestion in MNase digestion buffer (1x NEB MNase reaction buffer, 1.5 mM DTT, 5% PEG 6000). EDTA was added to a final concentration of 10 mM to stop the MNase digestion. Nuclei were lysed by adding Triton X-100 and sodium deoxycholate to a final concentration of 0.1% each, incubated on ice for 15 minutes and then vortexed. Soluble material containing mononucleosomes were recovered after centrifugation and subjected to immunoprecipitation. Sequencing libraries were prepared using a NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) and sequenced on an Illumina Hiseq 2500 to generate 50 bp paired-end reads.
NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
Mnase-digested chromatin input DNA
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| Data processing |
Raw, unaligned BAM files were first sorted with samtools sort -n and then converted to fastq files with bedtools bamtofastq.
Adapter trimming was performed using TrimGalore version 0.4.1.
Reads were mapped to the Arabidopsis genome (TAIR10) using Bowtie2 version 2.1.0.
Reads were filtered for a MAPQ score > 10 using SAMtools version 1.3 and subsequently filtered for duplicate reads using Picard tools MarkDuplicates version 1.141.
Biological replicates were subsequently merged for downstream analysis after confirming high correlation among replicates.
For data visualization, log2 ratio bigwig coverage files relative to input were generated using the deepTools version 2.5.0.1 utility bamCoverage with a bin size of 10 bp.
Peaks were called using the MACS2 version 2.1.0 callpeak function using -f BAMPE --broad --broad-cutoff 0.1 -g 1.2e8
Genome_build: TAIR10 (Arabidopsis thaliana)
Supplementary_files_format_and_content: *.bw: Normalized log2 ratio coverage of histone marks relative to relevant input sample. Data is generated from merged replicates.
Supplementary_files_format_and_content: *.narrowPeak: MACS2 narrowPeaks generated from merged replicates.
Supplementary_files_format_and_content: *.broadPeak: MACS2 broadPeaks generated from merged replicates.
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| Submission date |
Sep 30, 2018 |
| Last update date |
Mar 17, 2020 |
| Contact name |
Michael Borg |
| E-mail(s) |
borgmichael@gmail.com
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| Organization name |
Gregor Mendel Institute
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| Lab |
Berger Lab
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| Street address |
Dr. Bohr-Gasse 3
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL17639 |
| Series (2) |
| GSE120664 |
Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin |
| GSE120669 |
Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin |
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| Relations |
| BioSample |
SAMN10147965 |
| SRA |
SRX4779411 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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