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| Status |
Public on Sep 30, 2019 |
| Title |
H3K79me2_LIF |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14 ES cells
treatment: LIF
chip antibody: H3K79me2 (Active Motif 39143)
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| Treatment protocol |
E14 mESC cells were cultured in the presence of leukemia inhibitory factor (LIF) in order to maintain the pluripotent state of the cells. To exit from pluripotency and push the cells towards differentiation, LIF was withdrawn and retinoic acid (RA, Sigma, R2625) was added to the medium to a final concentration of 1 µM. For all experiments, 4h prior to harvest, cell culture medium was removed, cells washed twice with PBS and fresh ES medium was added containing either LIF or RA.
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| Growth protocol |
E14 mouse embryonic stem cells (mESCs) were cultured under feeder-free conditions and routinely passaged every two days in ES medium plus LIF: Glasgow Minimum Essential Medium (Sigma-Aldrich) supplemented with 17% FBS (HycloneTM, SV30160.03, GE Healthcare), 2 mM GlutaMAXTM (Gibco), 100 U/ml Penicillin-Streptomycin (Gibco), 1x MEM Non-Essential Amino Acid Solution (Gibco), 1 mM Sodium Pyruvate (Gibco), 0.5 mM 2-Mercaptoethanol (Gibco), and recombinantly expressed LIF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
6x105 low passage (< 10) E14 cells were plated per 15 cm culture dish and cultivated for 48 h in regular ES medium containing LIF. 4 h prior to cross-link cells were treated with LIF or RA as described in the treatment protocol. Then cells were washed with PBS, trypsinized and gently but thoroughly resuspended in ES medium to obtain single cell suspension. Cells were cross-linked adding 1% formaldehyde for 5 min and the reaction was quenched using 125 mM Glycin for another 5 min. After two wash steps with PBS, cell pellets were resuspended in 100 µl sonication buffer (20 mM Tris-HCl pH 7.5, 2 mM EDTA pH 8.0, 1% Triton X-100, 150 mM NaCl, 0.1% SDS and Complete proteinase inhibitor cocktail (Roche)) per 106 cells and chromatin was sheared by sonication applying a Bioruptor Pico (Diagenode) device for 25-35 cycles. Automatic ChIP was performed using the SX-8G Compact IP-Star liquid handler (Diagenode) in combination with Auto Histone Kits (Diagenode, C01010022). Using the pre-programmed method ‘indirect ChIP’, ChIP reactions were carried out in a final volume of 200 μl for 10 h followed by 5 h beads incubation and 5 min washes (at 4°C). After ChIP, eluates were recovered, RNase A-treated, decrosslinked ON at 65°C and treated with Proteinase K for 4 h at 55°C. The recovered DNA was purified using ChIP DNA Clean & Concentrator Kit (Zymo research, D5205).
Sequencing libraries were prepared using the NEBNext® UltraTM DNA Library Prep Kit (NEB, E7370) according to manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
RNA-seq: Raw sequencing reads were mapped to mm10 reference genome using Burrows-Wheeler Alignment tool (BWA-MEM algorithm, PMID: 19451168), processing fastq to bam files. Mapped reads were sorted using SAMtools (PMID: 19505943) and DESeq2 (PMID: 25516281) was applied for differential gene expression analysis using ENSEMBL gene annotation (Mus_musculus.GRCm38.90).
ChIP-seq: Each ChIP library was sequenced in two separate sequencing runs. Raw sequencing reads were mapped to mm10 reference genome using STAR Alignment tool (STAR_2.5.1b_modified, PMID: 23104886), allowing a maximum intron size of 1 to adjust for non-spliced input data. Subsequently reads from the same library were merged into one file.
ATAC-seq: We processed fastq to bam files using the Burrows-Wheeler Alignment tool (BWA) (PMID: 19451168) for mapping and SAMtools (PMID: 19505943) for filtering, sorting and removing duplicates.
Genome_build: mm10
Supplementary_files_format_and_content: RNA-seq: The DESeq2 derived final data file contains all genes with an adjusted p-value below 0.1 (padj < 0.1).
Supplementary_files_format_and_content: ChIP-seq: Mapped reads were converted to bigwig coverage tracks using the bam2bigwig script (https://github.com/milospjanic/bam2bigwig).
Supplementary_files_format_and_content: ATAC-seq: We called peaks on the alignment results using MACS2 (PMID: 18798982) and converted bam files to bigwig format using the bamToBigWig tool from the bioinformatics library Gonetics (P. Benner. Gonetics. https://github.com/pbenner/gonetics).
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| Submission date |
Sep 24, 2018 |
| Last update date |
Sep 30, 2019 |
| Contact name |
Laura V Glaser |
| E-mail(s) |
glaser@molgen.mpg.de
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| Phone |
+49 30 8413-1560
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Computational Molecular Biology
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| Street address |
Ihnestraße 63-73
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE120376 |
Genome-wide histone modification and accessibility maps of mESCs, in the pluripotent state and after induced differentiation |
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| Relations |
| BioSample |
SAMN10114372 |
| SRA |
SRX4733383 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3399481_E14_H3K79me2_LIF.bw |
484.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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