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| Status |
Public on Apr 30, 2019 |
| Title |
4C-seq_Enhancer1_CRISPR-clone_Rep2 |
| Sample type |
SRA |
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| Source name |
Mouse Embryonic Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
background strain: F1
cell type: Mouse Embryonic Stem Cells
treatment: 2i_Day3
chip antibody: Enhancer1
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| Growth protocol |
E14Tg2a or F1-hybride ESCs were cultured in standard culture medium on gelatin coated-dishes without feeder cells. Serum supplemented medium contains Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, L-glutamine (2 nM), Na-Pyruvate (1 mM), non-essential amino acids (0.1 mM each), Penicillin Streptomycin, 2-mercaptoethanol (55 µM) and LIF (1000 U/ml, Milipore). For serum free culture, cells were maintained in NDiff 227 (StemCells, Inc.) supplemented with MEK inhibitor PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) and LIF (1000 U/ml, Milipore). For EpiLSC-differentiation, 2i-ESCs were seeded in ~15,000 cells per cm2 density on Fibronectin (10 μg/ml) coated dishes and in NDiff 227 (StemCells, Inc.) supplemented with Penicillin Streptomycin (Gibco), 20 ng/ml ActivinA (R&D systems), 12 ng/ml bFGF (R&D systems) and 1% knock out serum replacement (Gibco) and cells were maintained for 72h in this culture.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 10 million cells were cross-linked for 10 minutes with 2% paraformaldehyde in ES-culture medium, quenched with glycine and lysed in 30 ml lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 0.2% NP-40, 1X protease inhibitors) for 30 minutes at 4°C . Nuclei were then incubated with 0.25% SDS for 30 minutes in NEB buffer3 followed by Triton X-100 treatment for 30 minutes both at 37°C. Nuclei were then digested with 700 units DpnII enzyme (NEB) overnight at 37 ̊C. Enzyme was inactivated at 65 ̊C for 15 min followed by ‘in nuclei’ ligation at 16 ̊C with 2,000 units T4 ligase (NEB)66. Samples were then treated with protease K and RNAseA and DNA was purified by phenol–chloroform (Sigma: 77618-500ml). DNA was further digested with 50 units BfaI, purified with QIAquick PCR purification columns followed by second ligation at 16°C. Next, 1000 ng of 4C-seq library was amplified with bait-specific inverse primers using Expand Long template PCR sytem (Roche 11759060001) for 28 PCR cycles.
PCR products were then purified and 50 ng DNA was used for library preparation using KAPA Hyperprep kit (Roche 07962363001) and 5 PCR cycles. Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and were size selected using bead purification protocol (200bp-1500bp). Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina Nextseq500 machines and generated 50bp pair end reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Library strategy: 4C-seq
4C-seq experiments were performed as described previously65 with minor modifications. Briefly, 10 million cells were cross-linked for 10 minutes with 2% paraformaldehyde in ES-culture medium, quenched with glycine and lysed in 30 ml lysis buffer (10mM Tris pH 7.5, 10mM NaCl, 0.2% NP-40, 1X protease inhibitors) for 30 minutes at 4 ̊ C . Nuclei were then incubated with 0.25% SDS for 30min in NEB buffer3 followed Triton X-100 treatment for 30 min both at 37 ̊ C. Nuclei were then digested with 700U DpnII enzyme (NEB) over night at 37 ̊C. Enzyme was inactivated at 65 ̊C for 15 min followed by ‘in nuclei’ ligation at 16 ̊C with 2000U T4 ligase (NEB)66. Samples were then treated with protease K and RNAse A and DNA was purified by phenol–chloroform (Sigma: 77618-500ml). DNA was further digested with 50U BfaI enzyme, purified with QIAquick PCR Purification columns followed by second ligation at 16 ̊ C. 1000ng of 4C-seq library was amplified with bait-specific inverse primers using Expand Long template PCR sytem (Roche 11759060001) for 28 PCR cycles (Table S1). PCR products were then purified and 50ng DNA was used for library preparation using KAPA Hyperprep kit (Roche 07962363001) and 5 PCR cycles. Libraries were sequenced on the Illumina NextSeq500 to obtain paired-end sequenced of 50bp-long reads
Genome_build: mm9
Supplementary_files_format_and_content: BigWig tracks
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| Submission date |
Mar 11, 2019 |
| Last update date |
May 01, 2019 |
| Contact name |
Hendrik G Stunnenberg |
| E-mail(s) |
[email protected]
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| Organization name |
Radboud University
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| Street address |
Geert Grooteplein 30
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| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE92412 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. |
| GSE113750 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (4C-seq) |
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| Relations |
| BioSample |
SAMN11105972 |
| SRA |
SRX5507855 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3665792_C2_rep2.bedGraph.gz |
185.7 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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