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| Status |
Public on Nov 26, 2018 |
| Title |
Patinet 2 ChIPseq K36me3Rx ChIP |
| Sample type |
SRA |
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| Source name |
primary fibroblast cell line
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| Organism |
Homo sapiens |
| Characteristics |
disease state: microcephalic dwarfism
cell line: primary fibroblast cell line
genotype: DNMT3A W330R heterozygous
spike-in: crosslinked Drosophila chromatin (S2 cells)
chip antibody: H3K36me3 (Active Motif; #61101)
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| Treatment protocol |
N/A
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| Growth protocol |
Fibroblast cell lines were maintained at 3% O2 in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies) supplemented with 10% FBS and 5% penicillin-streptomycin antibiotics or in AmnioMAX-C100 (Life Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked chromatin immunoprecipitation was adapted from a previous publication (Illingworth et al, PMID: 27723457) with the following modifications. 2x106 cells were harvested, washed and crosslinked with 1% methanol-free formaldehyde in PBS. Crosslinked cells were lysed directly for 15 min on ice in cold lysis buffer (10 mM EDTA, 50 mM Tris-HCl (pH8), 1% SDS). All buffers used during chromatin capture were freshly supplemented with protease inhibitors (Roche) and 1 mM DTT. For ChIP-Rx crosslinked Drosophila chromatin (S2 cells) was spiked into samples before sonication (ratio of 20:1 human to Drosophila cells). Lysates were sonicated with two 30 sec pulses using a soniprep 150 plus (MSE) followed by 10 cycles (30 s on / 30 s off on ‘high’ setting at 4°C) on a Bioruptor Plus (Diagenode). Following centrifugation for 10 min at 20,000xg, 4°C, supernatant was supplemented with Triton X-100 (final concentration 1%) and 50 μg/ml BSA and then added to Protein A dynabeads (Invitrogen) pre-coupled with the respective antibody (H3K27me3 antibody (Cell Signaling; C36B11; Cat.no. 9733S); H3K4me3 (Millipore; 07-473); H3K36me3 (Active Motif; #61101)). 10% of input material was retained for use as a reference. The bead-chromatin mixture was incubated under rotation at 4°C overnight. Beads were then washed twice for 10 min at 4°C with each of the following buffers: IP dilution buffer (2 mM EDTA, 150 mM NaCl, 20 mM Tris HCl pH8, 1% Triton X-100), buffer A’ (20 mM Tris pH8, 500 mM NaCl, 1 mM EDTA pH8, 1% TritonX-100, 0.1% Na-deoxycholate, 0.1% SDS), buffer B’ (20 mM Tris pH8, 1 mM EDTA pH8, 250 mM LiCl, 1% NP-40, 0.1% Na-deoxycholate). Finally, beads were washed three times with 1xTE buffer (1 mM EDTA pH8, 10 mM Tris pH8). Captured chromatin was eluted, followed by reverse crosslinking through addition of Tris HCl pH6.8 (100 mM), NaCl (300 mM) and 20 μg RNAse A at 65°C overnight. Input material was similarly de-crosslinked. Degradation of proteins with 20 μg Proteinase K and incubation at 37°C for 2 hrs. Samples purified with the MinElute PCR purification kit (QIAGEN).
ChIP-seq libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina (E7645) according to the manufacturer instructions using barcoded adapters (NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1 or 2), E7335; E7500S). For input reference libraries 50 ng material were used. Adapter197 ligated DNA was size selected for an insert size of 150 or 200 bp using AMPure XP beads. Libraries were amplified for 7 cycles in the final PCR amplification step. Amplified libraries were purified using AMPure XP beads and eluted in EB. Libraries were pooled in an equimolar ratios. For H3K27me3 single-end 50bp reads were generated on an Illumina HiSeq machine (GATC Biotech Konstanz, Germany). For H3K4me3, H3K36me3 ChIP-Rx and H3K27me3 ChIP-Rx single-end 75bp reads were generated on an Illumina NextSeq 550 machine.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Data processing |
ChIP-seq read quality was analyzed using FASTQC (v0.11.4), with low quality reads and adaptors removed using trim-galore with default settings (v0.4.1). Reads were aligned to the hg19 genome using bowtie 2 (v2.3.1, with settings: -N 1 -L 20 -- no-unal). Multi-mapping reads excluded using SAMtools (v1.6, with settings: -bq 10). PCR duplicates excluded using SAMBAMBA (v0.5.9). For ChIP Rx-seq, reads were aligned to combined hg19 and dm6 genomes using the same settings.
Tracks for data visualization generated using Homer (v4.8)73. Aligned BAMs were converted to tag directories setting the fragment length to 180bp and converted to bigWig files using Homer’s makeUCSCfile function after filtering with the removeOutOfBounds.pl function.
ChIP-seq peaks were called from tag directories using Homer’s findPeaks function (settings: -style histone) and the corresponding input chromatin control sample.
Genome_build: hg19, dm6
Supplementary_files_format_and_content: bigWig files derived as described above.
Supplementary_files_format_and_content: BED file of Homer peaks for ChIP samples.
Raw sequencing reads are available through EGA (protected patient data): EGAS00001003232
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| Submission date |
Sep 27, 2018 |
| Last update date |
Nov 26, 2018 |
| Contact name |
Duncan Sproul |
| Organization name |
University of Edinburgh
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| Department |
MRC Human Genetics Unit
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| Street address |
Crewe Road South
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| City |
Edinburgh |
| State/province |
Mid Lothian |
| ZIP/Postal code |
EH4 2XU |
| Country |
United Kingdom |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE120551 |
Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions (human fibroblast ChIP-seq) |
| GSE120558 |
Gain of function DNMT3A mutations cause microcephalic dwarfism and hypermethylation of Polycomb-regulated regions |
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| Relations |
| BioSample |
SAMN10136741 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3402911_P2_ChIPseq_K36me3Rx_ChIP.BED.gz |
482.1 Kb |
(ftp)(http) |
BED |
| GSM3402911_P2_ChIPseq_K36me3Rx_ChIP.bigWig |
479.8 Mb |
(ftp)(http) |
BIGWIG |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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