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Sample GSM1566967

Query DataSets for GSM1566967

Status

Public on Apr 08, 2016

Title

p35S:LFYTERE, rep2

Sample type

SRA

Source name

A. thaliana 15-day-old seedlings, p35S:LFYTERE

Organism

Arabidopsis thaliana

Characteristics

strain/background: CA29-17
genotype/variation: p35S:LFYTERE
tissue: seedlings
Stage: 15 days
chip antibody: rabbit (#4028) against the LFY C-terminal amino acids 223-424

Treatment protocol

No treatment.

Growth protocol

15-day-old p35S:LFY, p35S:LFYTERE and Col-0 (control) seedlings (complete seedlings, including aerial part+roots) grown under long day photoperiods at 23 °C on MS plates.

Extracted molecule

genomic DNA

Extraction protocol

The entire experiment from seed sowing through deep sequencing was performed twice to produce independent biological replicates. ChIP-seq was performed with an antibody raised in rabbit (#4028) against the LFY C-terminal amino acids 223-424 (BioGenes, Berlin, Germany). Briefly, 15-day-old 35S:LFY (pCA26) and 35S:LFYTERE (pCA29) and Col-0 (control) seedlings grown under long day photoperiods at 23 °C on MS plates were harvested and fixed as described previously. Frozen tissue was ground, filtered three times through Miracloth (Calibrochem), and washed as described previously thorough buffers M1, M2, and M3. Nuclear pellets were resuspended in sonic buffer as described (1 mM PEFA BLOC SC (Roche Diagnostics) was substituted for PMSF), split into technical duplicate samples, and sonicated with a Branson sonifier at continuous pulse (output level 3) for 8 rounds of 2 x 6 seconds and allowed to cool on ice between rounds. IP reactions were performed by incubating chromatin with 2.5 μl anti-LFY serum overnight at 4 °C as described. The immunoprotein-chromatin complexes were captured by incubating with protein A-agarose beads (Santa Cruz Biotechnology), followed by consecutive washes in IP buffer and then elution as described. Immunoprotein-DNA was then incubated consecutively in RNase A/T1 mix (Fermentas) and Proteinase K (Roche Diagnostics) as described, after which DNA was purified using Minelute columns (Qiagen). ChIP samples were tested for enrichment by QPCR.
Deep sequencing libraries were produced by standard Illumina protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer II

Description

Plant expressing a mutant version of Arabidopsis thaliana LFY cDNA (mutation T75E R112E) under the control of the 35S promoter.
Processed data file: peaks_p35SLFYTERE.bed

Data processing

Read mapping and duplicates removal were performed using the SHORE pipeline (preprocess, import, mapflowcell subprocesses) using -H 1,1 -M 0,4 -X 130 -B 1 as parameters. Peakfinding was performed with the shore subprogram using -B 1 as parameters. We kept as bound regions all peaks with a BH-FDR q < 10-7 in all 4 comparisons of the two replicates against the 2 controls. We obtained 1954 peaks for p35S:LFY and 176 for p35S:LFYTERE. Corresponding bed files with peak rank and CFR values are linked to the GEO Series record.
For each genomic position, the read number corresponds to the number of reads extended by 130 bp covering this position. For each bound region (ChIP-Seq peak), the coverage is defined as the area above the read count curve.
The normalization procedure was performed in two steps: 1) Intra genotype normalization between ChIP-seq replicates for the same genotype (Col-0, p35S:LFY or p35S:LFYTERE). For each genotype, we used the most significant peaks and plotted the coverage values of one replicate against the other. Replicates were then fused by calculating a normalized read count at each position. 2) Inter genotype normalization was performed based on the highest background peaks detected in the Col-0 control sample. For each background peak, coverages were compared between normalized p35S:LFY or p35S:LFYTERE and the Col-0 control sample and the regression coefficient was computed and used for final normalization for p35S:LFY and p35S:LFYTERE ChIP-seq.
The coverage fold reduction (CFR) due to the TERE mutation was computed : CFR = coverage(p35S :LFY)/coverage(p35S :LFYTERE)
Genome_build: TAIR10
Supplementary_files_format_and_content: Bed files with the peaks found by comparing p35S:LFY, or p35S :LFYTERE, and Col-0. Files contain position of the genome (TAIR10) and the rank of the bound regions (according to the FDR-BH q) and the CFR calculated by comparing the region in p35S:LFY and p35S:LFYTERE.

Submission date

Dec 16, 2014

Last update date

May 15, 2019

Contact name

Francois Parcy

E-mail(s)

francois.parcy@cea.fr

Organization name

CNRS

Lab

Physiologie Cellulaire et Végétale