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| Status |
Public on Oct 16, 2020 |
| Title |
H3K9me3_WT_6m_M_rep3 |
| Sample type |
SRA |
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| Source name |
Striatum
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| Organism |
Mus musculus |
| Characteristics |
tissue: Striatum
genotype: Wild-type
Sex: males
age: 6 months
replicats: rep3
chip antibody: H3K9me3: ab8898, Abcam
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| Treatment protocol |
Whole tissue: Frozen striata from 4 mice were pooled (sex-matched between HD and WT animals), cut into small fragments, fixed in 1% formaldehyde and incubated for 15 min at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M. Tissue fragments were washed with cold PBS supplemented with protease inhibitors. The tissues were then homogenized in sonication buffer, and lysates were sonicated to obtain DNA fragments <500 bp and centrifuged. The soluble chromatin fraction was pretreated with protein A Agarose/Salmon Sperm DNA (Millipore) for 1 h at 4°C. Subsequently, samples were incubated overnight at 4°C with antibodies to H3K27ac (Abcam, ab4729), H3K27me3 () or 7C2 RNAPII antibody. Protein A Agarose/Salmon Sperm DNA was then added and the mixture was incubated for 3 h at 4°C. Agarose beads were washed, protein-DNA complexes were eluted from the beads and decrosslinked, and DNA recovered by phenol chloroform extraction and ethanol precipitation after treatment with ribonuclease A (Abcam) and proteinase K.
NeuN sorted: Frozen striata from 4 mice were homogenized in ice-cold PBS supplemented with 1x Protease Inhibitors Cocktail (PIC, cOmplete EDTA free, Roche) and cross-linked in 1% formaldehyde for 15 minutes at room temperature. Cross-linking was stopped by the addition of glycine to final concentration 0.125 M and tissue was washed using ice-cold PBS. Cells were lysed then in Cell Lysis Buffer (10mM Hepes pH8; 85mM KCl; 0.5% NP-40) and nuclei were were collected after treatment with Nuclear Extraction Buffer (0.5% SDS, 10mM EDTA pH8, 50mM Tris). Purified nuclei were then resuspended in PBTB (PBS 1x, 5% BSA, 0.5% Tween-20) + 1x PIC, 3% Normal Horse Serum (NHS) and stained using α-NeuN antibody (1:1000, Merck Millipore). After washing, nuclei were labelled with α-Ms AF488 antibody (1:1500) and washed with ice-cold PBS. Immunostained nuclei were sorted using BD Aria Fusion flow cytometer, recovered in ice-cold 1x PBS, pelleted and stocked at -80ºC. Nuclei were resuspended in sonication buffer and sonicated to obtain DNA fragments <500 bp and centrifuged. The soluble chromatin fraction was pretreated with protein A Agarose/Salmon Sperm DNA (Millipore) for 1 h at 4°C. Subsequently, samples were incubated overnight at 4°C with antibodies to H3K27ac (Abcam, ab4729) or H3K27me3 (). Protein A Agarose/Salmon Sperm DNA was then added and the mixture was incubated for 3 h at 4°C. Agarose beads were washed, protein-DNA complexes were eluted from the beads and decrosslinked, and DNA recovered by phenol chloroform extraction and ethanol precipitation after treatment with ribonuclease A (Abcam) and proteinase K.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with Qubit (Invitrogen). ChIP-seq libraries were prepared from 2 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode), according to manufacturer's instructions. Illumina compatible indexes were added through PCR amplification (7 cycles). Amplified libraries were purified and size-selected using Agencourt® AMPure® XP beads (Beckman Coulter) to remove unincorporated primers and other reagents. Prior to analyses DNA libraries were checked for quality and quantified using a 2100 Bioanalyzer (Agilent). Libraries were sequenced on Illumina Hiseq 4000 sequencer as Paired-End 50 base reads following Illumina’s instructions. Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
ChIP is performed using the striatum of HD KI and control mice at 6 months. Striata from different animals are pooled. 3rd replicate using males
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| Data processing |
Image analysis and base calling were performed using RTA 2.7.3 and bcl2fastq 2.17.1.14.
Sequence reads were mapped to reference genome mm10 using Bowtie 1.0.0 with the following parameters -m 1 --strata --best -y -S -l 40 -p 2.
Genome_build: mm10
Supplementary_files_format_and_content: Wig files were generated using with an in-house script (Variable step, span=25, reads were elongated to 200b).
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| Submission date |
Feb 03, 2020 |
| Last update date |
Oct 16, 2020 |
| Contact name |
Jonathan Seguin |
| E-mail(s) |
seguin.jonathan@gmail.com
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| Phone |
+333688245252
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| Organization name |
UKBB-DBM
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| Lab |
group of Prof. Schwaller
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| Street address |
Spitalstrasse 33
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| City |
Basel |
| ZIP/Postal code |
CH-4056 |
| Country |
Switzerland |
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| Platform ID |
GPL21103 |
| Series (1) |
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| Relations |
| BioSample |
SAMN13976743 |
| SRA |
SRX7667042 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4293665_H3K9me3_WT_6m_M_rep3.wig.gz |
237.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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