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Sample GSM2786274 Query DataSets for GSM2786274
Status Public on Sep 12, 2018
Title Input chromatin caa39
Sample type SRA
 
Source name 6 day-old aerial parts
Organism Arabidopsis thaliana
Characteristics genotype/variation: caa39 mutant
antibodies: none
Growth protocol Seeds are stratified for 3 days at 4°C in darkness before transfer to growth chamber for 6 days (16hL/8hD: 8 am-12 pm, ~80 µE, ~65% humidity, ~21°C). Media: Murashige and Skoog (MS) 1/2, 1% sucrose, 0,4% phytagel or 0.8% Agar
Extracted molecule genomic DNA
Extraction protocol Two hundred 6 day-old seedlings were used per biological replicates. Seedlings were quickly harvested using a cat hair comb, root material was cut-off and the remaining photosynthetic material was fixed with 1% formaldehyde for 9 minutes. Crosslinking was quenched by addition of glycine at 125 mM final concentration and 5 minutes incubation, washed twice in ddH2O and the excess water was drained using kimwipes. Plant material was then ground in liquid nitrogen, resuspended in extraction buffer 1 (0.4M Sucrose, 10mM Tris pH8, 10 mM MgCl2, 5mM β-Mercaptoethanol and protease inhibitor), filtered with four layers of miracloth and centrifuged 20 minutes at 4000 rpm, 4°C. The pellet was resuspended in extraction buffer 2 (0.25M Sucrose, 1% Triton X100, 10mM Tris pH8, 10 mM MgCl2, 5mM β-Mercaptoethanol and protease inhibitor) and centrifuged 10 minutes, 13000 rpm, 4°C. The pellet was resuspended in 300 µL extraction buffer 2 and layered on top of 300 µL extraction buffer 3 (1.75M Sucrose, 0.15% Triton X100, 10mM Tris pH8, 2 mM MgCl2, 5mM β-Mercaptoethanol and protease inhibitor) and centrifuged at 13000 rpm for 1h, 4°C. The resulting pellet was resuspended in 130 µL of nuclear lysis buffer (50mM Tris pH8, 10 mM EDTA, 1% SDS and protease inhibitor) and sheared using a Bioruptor for 10 cycles, high settings, 30 sec ON/60 sec OFF, twice. Then, the sheared chromatin was diluted ten times in ChIP dilution buffer (1.1% Triton X100, 16.7mM Tris pH8, 1.2mM EDTA, 167mM NaCl and protease inhibitor). In parallel, 10 µL protein G Dynabeads® were washed twice in ChIP dilution buffer and incubated either with 2 µg of mouse IgG (Sigma), anti-H3K27me3, anti-H3K9m2, anti-MAT or anti-GFP during 2h at 4°C under gentle agitation. Antibody-conjugated beads were washed twice with 100 µL ChIP dilution buffer and 400 µL of input was submitted to IP for 4h or at 4°C under gentle agitation. Immunocomplexes were washed twice with each following buffer, 5 minutes, 4°C, gentle agitation, in that order: low salt wash buffer (150mM NaCl, 0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris pH8), high salt wash buffer (500mM NaCl, 0.1% SDS, 1% Triton X100, 2mM EDTA, 20mM Tris pH8), LiCl wash buffer (250mM LiCl, 1% Igepal CA-630, 1% sodium deoxycholate, 1mM EDTA, 10mM Tris pH8), TE buffer (10mM Tris pH8, 1mM EDTA). Immunocomplexes were then eluted twice with 100 µL elution buffer (100mM NaHCO3, 1% SDS), 15 minutes at 65°C, agitation. The 200 µL eluate was reversed crosslinked by adding 16 µL of 2.5M NaCl and incubating at 65°C overnight and DNA was purified using phenol/chloroform extraction. A 40 µL aliquot of input was reversed crosslinked and purified in parallel. DNA was quantified using Qubit® dsDNA HS assay kit (ThermoFischer Scientific).
TruSeq ChIP Library Preparation kit
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
Data processing Checking sequence quality with FASTQC (0.11.5)
Adaptator trimming with trimmomatic (0.36)
sequences were aligned with bowtie 2 (2.2.9) againt TAIR10.31 and filtered with bamtools filter (2.4.0) to remove sequence with a MapQuality below 20. Finally MarkDuplicate (2.8.1) was used to remove PCR duplicate
Peak calling with diffReps-nb (1.55.2)
BamCoverage (2.4.1) was used to create bigwig files for visualization
Genome_build: TAIR10-31
Supplementary_files_format_and_content: bigwig files, Excel file containing gene and TE transcript quantification in the mutant versus the wt
 
Submission date Sep 15, 2017
Last update date May 15, 2019
Contact name Christophe Laloi
E-mail(s) christophe.laloi@univ-amu.fr
Phone +334 91 82 95 64
Organization name Aix-Marseille Université
Department Biology
Lab LGBP
Street address 163 avenue de Luminy
City Marseille
State/province France
ZIP/Postal code 13009
Country France
 
Platform ID GPL19580
Series (2)
GSE103924 Topoisomerase VI participates to a chromatin barrier-like function that prevents heterochromatin spreading in euchromatic islands (HTS)
GSE129249 Topoisomerase VI participates to a chromatin barrier-like function that prevents heterochromatin spreading in euchromatic islands
Relations
BioSample SAMN07656235
SRA SRX3190308

Supplementary file Size Download File type/resource
GSM2786274_S001556_markduplicate20.bigwig 11.5 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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