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| Status |
Public on Aug 11, 2021 |
| Title |
ESC input |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: Embryonic stem cells
chip antibody: none
treatment: transfection with pRNA
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| Treatment protocol |
9 x 105 ESC were plated in gelatin-coated 10cm culture dish and transfected with 42 μg pRNA, or RNA-control using Lipofectamine MessengerMAX reagent (Invitrogen) in Opti-MEM GlutaMAX (Life Technologies) reduced-serum medium for 48h
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| Growth protocol |
E14 mouse embryonic stem cells were cultured in either 2i media composed of DMEM-F12 and Neurobasal medium (1:1, Life Technologies), supplemented with 1× N2/B27 (Life Technologies), 1× penicillin/streptomycin/l-glutamine (Life Technologies), 50 μM β-mercaptoethanol (Life Technologies), recombinant leukemia inhibitory factor, LIF (Polygene, 1,000 U/ml) and MEK and GSK3β inhibitors, 2i (Sigma CHIR99021 and PD0325901, 3 and 1 μM, respectively). ESCs were seeded at a density of 50,000 cells/cm2 in culture dishes (Corning® CellBIND® surface) coated with 0.1% gelatin without feeder layer. Propagation of cells was carried out every 2 days using enzymatic cell dissociation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed with lysis buffer (50mM Tris-HCl, pH 8.1, 10 mM EDTA, pH 8, 1% SDS, 1X protease inhibitor cOmplete EDTA-free cocktail, Roche). Nuclei were sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and incubated overnight with the ChIP-grade antibodies against H3K9me2 and H3K9me3. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR.
The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) was used in the following steps. ChIP samples (1 ng) was end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).
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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 |
Fastq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
Genome_build: mm10
Supplementary_files_format_and_content: bigwig
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| Submission date |
May 19, 2020 |
| Last update date |
Aug 11, 2021 |
| Contact name |
Raffaella Santoro |
| E-mail(s) |
raffaella.santoro@dmmd.uzh.ch
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| Phone |
+41 44 635 54 75
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| Organization name |
University of Zurich
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| Department |
Dep. of Molecular Mechanisms of Disease
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| Street address |
Winterthurerstrasse 190
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| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE150822 |
Identification of nucleolar associated domains (NADS) |
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| Relations |
| BioSample |
SAMN14970648 |
| SRA |
SRX8362339 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4559242_ESC_input_R1.norm.bw |
76.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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