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| Status |
Public on Nov 24, 2018 |
| Title |
2i-H3K27me3-rep2-spikeIn-13613 |
| Sample type |
SRA |
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| Source name |
Embryonic Stem Cells (ESCs)
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| Organism |
Mus musculus |
| Characteristics |
antibody: H3K27me3
cell type: ESCs
cell line: E14
genotype/variation: wild type
strain background: 129/Ola
treatment: Adapted to defined media containing 2i >14 days
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| Treatment protocol |
ESCs were adapted to defined media containing 2i between 14 and 28 days; Treatment with 5-azacytidine (Sigma) was performed for 6 days at a final concentration of 1 µM. 5-azacytidine was refreshed every day.
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| Growth protocol |
E14 ESCs (129/Ola background) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 15% fetal bovine serum, 5µM beta mercaptoethanol (BME) (Sigma) and Leukemia inhibitory factor (LIF; 1000U/ml) (Millipore), referred to as serum ESCs, or in serum-free N2B27 (or Ndiff) supplemented with PD0325901 (1 µM), CH99021 (3 µM) and LIF (1000U/ml), referred to as 2i ESCs. Serum+2i cells were grown in Serum medium supplemented with PD0325901 (1 µM) and CH99021 (3 µM). The Eed-/- and Suz12-/- ESCs originate from the lab of Anton Wutz (Schoeftner et al., 2006 and Wutz & Jaenisch, 2000) and Kristian Helin (Pasini et al., 2007), respectively
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For RNA-Seq, total RNA was isolated using a RNeasy mini kit (Invitrogen) according to manufacturer’s instructions with on-column DNAseI treatment. Ribosomal RNA was depleted using Ribo-Zero Gold rRNGA Removal Kit (Illumina) according to manufacturer protocol starting from 5µg RNA. The resulting 180uL of rRNA depleted RNA was purified by mixing 18uL 3 M Sodium Acetate and 2 uL of 20 mg/mL glycogen, then adding 3x volumes of ice-cold 100% ethanol and incubation at -20°C for 1 hour. Samples were centrifuged at 10.000g for 30 minutes. After removal of supernatant, RNA pellet was washed in 500uL ice-cold 70% ethanol and centrifuged at 10.000g. Supernatant was removed and RNA was eluted in 40uL nuclease free H2O. rRNA depleted RNA was fragmented to ~200bp size using 10uL of a 5x 200mM Tris-acetate, 500 mM Potassium Acetate, 150 mM Magnesium Acetate, pH 8.2 solution at 95°C for 200 seconds, then incubated on ice for 10 minutes before repeating the ethanol purification procedure. First strand cDNA synthesis was performed using SuperScript III RT enzyme (Life Technologies), purified using Qiaquick MinElute Column (Qiagen). For ChIP-Seq, chromatin extracts were prepared by on-plate cell crosslinking in 1% PFA for 8 minutes. Crosslinking was quenched using 125 mM freshly dissolved Glycine final concentration. Fixed cells were washed in PBS twice, then collected by scraping. Pellets were lysed and sonicated in 50 mM Tris pH 8.0, 1% SDS and fresh protease inhibitor cocktail (Roche) at a density of 15 million cells per mL. The cells were sonicated in a Diagenode Bioruptor Pico for 8-10 30-second cycles. Proper sonication was verified using agarose gel size checks after decrosslinking and the eluted DNA was quantified using Qubit HS. 25ug DNA equivalent mouse chromatin was ChIPped using 4ug H3K27me3 (Millipore 04-779) or 2ug H3K36me3 (Diagenode pAb-192-050) antibody. Spike-in normalization was performed by adding 50ng Drosophila Melanogaster material (Active Motif 53083) and 2ug Drosophila-specific H2Av antibody (Active Motif 61686) to the mouse chromatin. ChIPs were diluted 9-fold using IP buffer (1% Triton X100, 1.2mM EDTA, 16.7mM Tris pH8.0, 167mM NaCl) and incubated overnight at 4°C while rotating. Subsequently, a mixture of 10uL Protein A and 10uL protein G (thermo) was blocked twice in IP buffer with 0.15% SDS and added to the ChIP. ChIPs with beads were left to incubate for 1 hour and precipitated using a magnetic rack. Chromatin was washed once (2mM EDTA, 20mM Tris pH8.0, 1% Triton, 0.1% SDS, 150mM NaCl), twice (2mM EDTA, 20mM Tris pH8.0, 1% Triton, 0.1% SDS, 500mM NaCl) and twice (1mM EDTA, 10 mM Tris pH8,0). Specific ChIPped DNA was eluted using 200mM NaCl, 1%SDS, 20mM Tris pH8.0 by shaking at 65°C for one hour, addition of and 1:100 10mg/mL Proteinase K and shaking at 55°C for one hour, then shaking at 65°C for at least four hours. The DNA was purified using Qiagen Minelute according to manufacturer instructions. For Bisulfite-seq, genomic DNA was extracted using the Wizard genomic DNA isolation kit (Promega). 200ng of purified genomic DNA was digested with Proteinase K before proceeding with sample processing for WGBS.
For RNA-Seq, second strand synthesis was performed using incorporation of dUNTP’s. Libraries were generated using Kapa Hyper Prep kit (KAPA Biosystems) according to manufacturer’s protocol with modification of dUNTP cleavage before amplification. Libraries were size selected using Agencourt Ampure XP beads (Beckman Coulter). For ChIP-Seq, libraries were generated using Kapa Hyper Prep on 5ng DNA according to manufacturer instructions and size selected using Ampure XP beads. DNA was quantified using Qubit HS and DNA fragment sizes checked using Agilent Bioanalyzer HS. For WGBS, sample prep, paired-end sequencing and further analysis of Bisulfite-seq/WGBS was performed as in (Farlik et al., 2015).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP targeting H3K27me3 modification using antibody H3K27me3 (Millipore 04-779)
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| Data processing |
For RNA-Seq and ChIP-Seq, Initial data processing and base calling was performed using the Illumina Analysis Pipeline. Initial data processing and base calling of Bisulfite-seq/WGBS was performed as in (Farlik et al., 2015).
For RNA-Seq, strand specific RNA-seq reads were aligned to the mm9 reference genome and counted using STAR v2.5.2b with parameters --quantMode GeneCounts --outSAMmapqUnique 255 --outSAMmultNmax 1 --outFilterMismatchNoverLmax 0.05 --outFilterMatchNmin 16
For ChIP-Seq, reads were mapped to a merged mm9 and dm3 genome using bowtie2 2.0.2 with parameters --very-sensitive --end-to-end. Duplicate and unmapped reads were removed. The data mapped to dm3 and mm9 genomes were split into separate files.
For RNA-Seq, only uniquely mapping tags (mapq=255) were kept. Bigwigs were generated using bam2bw (https://bitbucket.org/simonvh/bam2bw.git) v1.25 with parameters –e 150 –c –p.
For ChIP-Seq, Only uniquely mapping tags (mapq >=30) were kept for quantification. Bigwigs were generated using bam2bw (https://bitbucket.org/simonvh/bam2bw.git) v1.25 with parameter –e 180 and custom scaling factor -s set to account for the relative abundance of the dm3 spike-in chromatin.
Data processing and data analysis of Bisulfite-seq/WGBS data was performed as in Habibi et al. (2013)
Genome_build: mm9 + dm3
Supplementary_files_format_and_content: bigWig files are directly compatible with the UCSC genome browser.
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| Submission date |
Apr 04, 2018 |
| Last update date |
Nov 25, 2018 |
| Contact name |
Hendrik Marks |
| E-mail(s) |
h.marks@ncmls.ru.nl
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| Organization name |
Radboud University Nijmegen, RIMLS
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| Department |
Molecular Biology
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| Street address |
Geert Grooteplein 26/28
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE101675 |
Integrative Proteomic Profiling Reveals PRC2-Dependent Epigenetic Crosstalk Maintains Ground-State Pluripotency. |
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| Relations |
| BioSample |
SAMN08863976 |
| SRA |
SRX3883731 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3080982_2i-H3K27me3-rep2-spikeIn-13613.bw |
289.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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