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| Status |
Public on Jan 23, 2023 |
| Title |
Input_EpiSC_WT_Rep1 |
| Sample type |
SRA |
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| Source name |
Mouse Epiblast Stem Cells
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| Organism |
Mus musculus |
| Characteristics |
strain background: 129/Ola
genotype: Wild type (WT)
cell type: ES-E14 derived EpiSC
Stage: EpiSC
antibody: Input
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| Growth protocol |
E14 mouse WT and mutant (Dnmt3a -/- and Dnmt3b -/-) ESCs were cultured in high-glucose DMEM (Euroclone) supplemented with 15% FBS (Millipore Corp., Billerica, MA, USA), 0.1 mmol/l nonessential amino acids (Invitrogen), 1 mmol/l sodium pyruvate (Invitrogen), 0.1 mmol/l β-mercaptoethanol, 1500 U/ml Leukemia Inhibitory Factor (LIF; Millipore), 25 U/ml penicillin, and 25 μg/ml streptomycin. For EpiSC induction, a single-cell suspension was seeded onto Geltrex (A1413202 GIBCO)-coated plates at a density of 10,000 cells cm-2 in N2B27 medium supplemented with 20ng/ml ActivinA (PHC9564 GIBCO) and 12 ng/ml bFGF (PHG0026 GIBCO). The cells were passaged 1:3 as small clumps using Collagenase IV (17104019 GIBCO). EpiSCs were collected for DNA and RNA analyses after 14 days of induction followed by daily medium changes. N2B27 medium is composed by 50% advanced DMEM/F12 (12634028 GIBCO) and 50% Neurobasal medium (21103049 GIBCO), supplemented with 0.5% N2 Supplement (17502048 GIBCO), 1% B27 Supplement (17504044 GIBCO), 0.033% BSA solution (A9647 SIGMA), 50 uM β-mercaptoethanol (M3148 Sigma), 2mM Glutamax (35050038 GIBCO), 100U/ml penicillin and 100 ug/ml streptomycin (DE17-602E LONZA). For Meso-endoderm directed lineage specific differentiation, EpiSCs were plated as small clumps onto Geltrex-coated plates in EpiSCs medium for 24 hours. The day after, medium was replaced with N2NB27 medium consisted of 50% advanced DMEM/F12 (12634028 GIBCO) and 50% Neurobasal medium (21103049 GIBCO), supplemented with 0.5% N2 Supplement (17502048 GIBCO), 1% B27 supplement minus Vitamin A (12587010 GIBCO) and 3 uM iGSK3β (CHIR99021 SIGMA). Cells were fed daily until the end of differentiation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 2*10^7 cells were cross-linked by addition of formaldehyde to 1% for 10 min at RT, quenched with 0.125 M glycine for 5 min at RT, and then washed twice in cold PBS. The cells were resuspended in Lysis Buffer 1 (50 mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100 and protease inhibitor) to disrupt the cell membrane and in Lysis Buffer 2 (10 mM Tris-HCl pH8.0, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA and protease inhibitor) to isolate nuclei. The isolated nuclei were then resuspended in SDS ChIP Buffer (20 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and protease inhibitors). Extracts were sonicated using the BioruptorH Twin (Diagenode) for 2 runs of 10 cycles [30 sec ‘‘ON’’, 30 sec ‘‘OFF’’] at high power setting. Cell lysate was centrifuged at 12,000 g for 10 min at 4°C. The supernatant was diluted with ChIP Dilution Buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton) before immunoprecipitation step. Streptavidin beads (Dynabeads®Protein G, Life Technologies) were saturated with PBS/1% BSA and the samples were incubated with 2 ug of antibody overnight at 4°C on a rotator. Next day samples were incubated with saturated beads for two hours at 4°C on a rotator. Successively immunoprecipitated complexes were washed five times with RIPA buffer (50 mM Hepes-KOH pH7.6, 500mM LiCl, 1mM EDTA, 1% NP-40, 0,7% Na-Deoxycholate) at 4°C for 5 minutes each on a rotator. Elution Buffer was added and incubated at 65°C for 15 minutes. The decrosslinking was performed at 65°C overnight. De-crosslinked DNA was purified using QIAQuick PCR Purification Kit (QIAGEN) according to the manufacturer’s instruction.
Starting from 10 ng of ChIP eluted sample, the library was produced for the genome wide analysis following the NEBNext® ChIP-Seq Library Prep Reagent Set for Illumina® (E 6240L NEB) manufacturer’s instructions. Libraries were sequenced on Illumina NextSeq 500 System (single-end 75 bp reads).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Following quality controls (performed with FastQC v0.11.2), sequencing reads were aligned to mouse reference genome (mm10/GRCm38) using Bowtie v2.3.4.1 (options: -q --local). Duplicated alignments (identified by Picard MarkDuplicates, https://broadinstitute.github.io/picard) and low-quality alignments/multi-mapping reads were excluded using SAMtools (command: samtools view –F1804 –q 30). Coverage tracks were generated from filtered alignments using the deepTools bamCoverage utility. IP and corresponding control (Input DNA) datasets were treated identically. Peak calling was performed using MACS v2.1.1 (options: callpeak -t=<IP> -c=<Input> -g mm --nomodel –extsize=<ES> --broad –q 0.05 --broad-cutoff 0.05 --fe-cutoff 1). The read extension size (ES) was estimated by cross-correlation using the phantompeakqualtools package. Input-normalized ChIP-seq signals were obtained using the deepTools bamCompare utility (options: --extendReads=<ES> --scaleFactorsMethod readCount --binSize 10 --operation log2). These processing steps were applied to all sample groups.
Genome_build: mm10/GRCm38
Supplementary_files_format_and_content: BigWig files containing ChIP-seq read densities for IP and Input DNA control (RPKM unit).
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| Submission date |
Mar 07, 2021 |
| Last update date |
Jan 23, 2023 |
| Contact name |
Andrea Lauria |
| E-mail(s) |
andrea.lauria@unito.it
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| Organization name |
University of Turin
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| Department |
Life Sciences and Systems Biology
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| Lab |
Functional Genomics, Epigenomics - S. Oliviero
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| Street address |
Via Accademia Albertina, 13
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| City |
Turin |
| ZIP/Postal code |
10123 |
| Country |
Italy |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE168411 |
DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells [ChIP-seq] |
| GSE168415 |
DNMT3B supports meso-endoderm differentiation from mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN18201674 |
| SRA |
SRX10254195 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5138720_Input_EpiSC_WT.filt.bw |
108.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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