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| Status |
Public on Dec 06, 2016 |
| Title |
PLT2_IP1 |
| Sample type |
SRA |
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| Source name |
roots
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: plt1plt2 double mutant
tissue type: roots
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| Treatment protocol |
PBS-S medium was complemented with 1% (V/V) formaldehyde (16% methanol-free formaldehyde, Polysciences). A vacuum pump was used to reduce the pressure to ~0.2 atm for 3 times within 5 min. Root tips were left to cross link protein-DNA complexes for an additional 2x 5min. Cross linking was stopped by the addition of 2M glycine to a final concentration of 0.125M followed by an incubation for 10 min. Root material was washed 3 times in PBS-S and stored at -80°C. ChIP was performed as described by Song, J., Rutjens, B. & Dean, C. in Plant Epigenetics and Epigenomics Vol. 1112 Methods in Molecular Biology (eds Charles Spillane & Peter C. McKeown) Ch. 11, 165-175 (Humana Press, 2014), with the following modifications: Ground samples were thawed in extraction buffer 1 and passed through a double layer of Miracloth and pelleted. The nuclei-enriched pellet was treated twice with extraction buffer 2. After the second spin the pellet was resuspended in 200 μl NLB and sonicated in a Bioruptor (Diagenode) for 2x 5min on low and 1x 5min on medium energy. In between sonication sessions the water was cooled with ice. 5 μl anti-GFP antibody (AB290, Abcam) was used to coat 15 μl Protein-A Dynabeads (Invitrogen) in CDB for 2h at room temperature. Diluted ChIP samples and antibody coated Dynabeads were incubated O/N at 4°C on a rotator. Beads were washed 2x with LSB, 1x HSB and 2x TE. Beads were incubated with 100 µl TE+1%SDS at 65°C on an Eppendorf Thermomix (800 rpm) for 15 min upon which the isolated DNA was purified using Zymo Research ChIP DNA clean-up (according to the manufacturer’s instructions).
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| Growth protocol |
Five ml of dry Arabidopsis seeds (PTL2::PLT2-YFP) were sterilized with chlorine gas for 8h and stratified in 10ml sterile MQ water for 48h. Seeds were transferred to 0.5x GM agar plates topped with a nylon mesh (Sefar Nitex 03-100/44) and grown for 4-5 days (~120 plates/ChIP). Root tips (~5mm) were cut and transferred to PBS-S (PBS + 0.4M Sucrose).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Two replicate ChIP samples and one input sample were sequenced. Sequencing library preparation and subsequent Solid Sequencing were performed as described in Mokry, M. et al. Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles. PloS one 5, e15092.
According to Mokry, M. et al. Efficient Double Fragmentation ChIP-seq Provides Nucleotide Resolution Protein-DNA Binding Profiles. PloS one 5, e15092
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 3.0 |
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| Description |
PLT2_IP1_IP2_pooled.wig
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| Data processing |
CSFASTQ files were extracted from the XSQ file using the XSQConverter tool developed by Wim Spee in the Cuppen group
Quality checking and filtering was performed with the script SOLiD_preprocess_filter_v2.pl written by Ariella Sasson, Rutgers University (https://github.com/fls-bioinformatics-core/HTS_SOLiD_Preprocessing) with the following parameters: SOLiD_preprocess_filter_v2.pl -f IP1.csfasta -g IP1_F3_QV.qual -p 3 -q 22 -e 10 -d 9 -n on -t on -u 25 -a on -v on -o IP1
CSFASTA and QUAL files were converted back to CSFASTQ files using the script qualfa2fq.pl of bwa version 0.5.9
CSFASTQ files were converted to encoded FASTQ files with the script solid2fastq_v2.pl
reads were mapped on the Arabidopsis thaliana TAIR10 genome release with the command bwa samse version 0.5.9 and default parameter settings
the SAM file was converted into a BAM file with SAMtools version 0.1.19-44428cd
only uniquely mapped reads were retained and BAM files were converted into CSAR input file format as follows: samtools view -b -F 1548 -q 30 IP1.bam | bamToBed -i stdin | awk '{print 1 "\t" $3-$2 "\t" $6 "\t" $1 "\t" $2+1}' | gzip -c > IP1.csar.gz
bamToBed is from BEDtools version 2.17.0
To maximize the IP signal, reads from the two IP samples, IP1 and IP2, were pooled as follows. Only one read per position was considered: gzcat PLT2_IP1_IP2.pooled.rmdup.csar.gz | sort -k4,5 -n -r | uniq | gzip -c > PLT2_IP1_IP2.rmdup.csar.gz
The fragment length estimated by the R package spp 18 for the pooled IP1 and IP2 samples was 130 bp
Peaks were called using CSAR version 1.10.0. The fragment length was provided as estimated, using the parameter w in the function mappedReads2Nhits. Ten iterations of the function permutatedWinScores were used to estimate the False Discovery Rate. To account for the pooling of the two replicate IPs, a stringent FDR threshold of 0.01 was used to retain significant peaks. The other parameter settings were maintained as default.
Genome_build: TAIR10
Supplementary_files_format_and_content: The wig file was generated using CSAR; Scores represent CSAR scores.
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| Submission date |
Mar 31, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Luca Santuari |
| Organization name |
Wageningen University
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| Department |
Plant Sciences Group
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| Lab |
Plant Developmental Biology
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| Street address |
Droevendaalsesteeg 1
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| City |
Wageningen |
| ZIP/Postal code |
6708PB |
| Country |
Netherlands |
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| Platform ID |
GPL10088 |
| Series (2) |
| GSE79754 |
Chromatin immunoprecipitation and sequencing of pPLT2::PLT2-YFP in the root of plt1plt2 double mutant |
| GSE79755 |
The PLETHORA gene regulatory network guides growth and cell differentiation in Arabidopsis roots |
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| Relations |
| BioSample |
SAMN04592581 |
| SRA |
SRX1671294 |
| 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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