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| Status |
Public on Sep 12, 2018 |
| Title |
H3K27me3 caa39 2 |
| Sample type |
SRA |
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| Source name |
6 day-old aerial parts
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| Organism |
Arabidopsis thaliana |
| Characteristics |
genotype/variation: caa39 mutant
antibodies: H3K27me3 (abcam ab6002)
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| 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
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| 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
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| 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
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| Submission date |
Sep 15, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Christophe Laloi |
| E-mail(s) |
christophe.laloi@univ-amu.fr
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| Phone |
+334 91 82 95 64
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| Organization name |
Aix-Marseille Université
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| Department |
Biology
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| Lab |
LGBP
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| Street address |
163 avenue de Luminy
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| City |
Marseille |
| State/province |
France |
| ZIP/Postal code |
13009 |
| Country |
France |
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| 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 |
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| Relations |
| BioSample |
SAMN07656237 |
| SRA |
SRX3190305 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2786272_S001618_markduplicate20.bigwig |
7.2 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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