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| Status |
Public on Mar 02, 2020 |
| Title |
Oct4_Tet1KO_EpiLC_Tet1tm1Koh_rep2 |
| Sample type |
SRA |
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| Source name |
Tet1tm1Koh EpiLC
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| Organism |
Mus musculus |
| Characteristics |
cell type: EpiLC
genotype: Tet1_KO
strain: C57BL/6J x 129S6 F1 hybrid
chip antibody: Oct4
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| Growth protocol |
Differentiation toward EpiLCs was performed as previously described but without activin A supplementation (Buecker et al. 2014). Briefly, feeder-depleted ESCs were first adapted at least 5 passages in defined media referred as 2iL media, which is composed of N2B27 basal media supplemented with 1 µM MEK inhibitor PD0325901 (Cat#Axon 1386, Axon Medchem BV), 3 µM GSK3 β inhibitor CHIR99021 (Cat#Axon 1408, Axon Medchem BV) and 1000 U/ml ESGRO LIF (Cat#ESG1107, Millipore). The N2B27 basal media is prepared as a 1:1 mixture of DMEM-F12 (Cat#11320-074, Invitrogen) and Neurobasal medium (Cat#21103-049, Invitrogen) supplemented with 1 x N2 (Cat#17502-048, Invitrogen), 1 x B27 (Cat#17504-044, Invitrogen), 1 mM L-glutamine, 0.1 mM 2-mercaptoethanol and 100:100 units:g/ml penicillin:streptomycin. 2iL-adapted ESCs were seeded at 2-3 x 105 cells per 10-cm2 on dishes coated with 5 g/10 cm2 fibronectin and differentiated for 2 days in N2B27 basal media supplemented with 1% knockout serum replacement KSR (Cat#10828-028, Invitrogen) and 12 ng/ml bFGF (Cat#100-18C, Peprotech).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were trypsinized and chemically cross-linked with 1% methanol-free formaldehyde (Cat#04018, Polysciences) for 10 min at room temperature, then quenched with 0.125 M glycine. Fixed cells were lysed sequentially in lysis buffer I (50 mM HEPES-KOH pH 7.5, 140 mM NaCl, 0.25% Triton X-100, 0.5% NP-40, 10% glycerol, 1 mM EDTA), lysis buffer II (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA), followed by lysis buffer III (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine). Chromatin fractions were sheared to 200–500 bp using Bioruptor® Plus sonicator under high power setting for 20 cycles (30 seconds ON, 30 seconds OFF). The sheared DNA was measured on an Agilent 2100 Bioanalyzer using High Sensitivity DNA Analysis Kits (Agilent, Cat#5067-4626). The high-size fragments (> 500 bp), if detected, was then removed with size-selection using Agencourt AMPure XP beads (Cat#A63881, Beckman Coulter). The sheared DNA then was incubated using appropriate antibodies (and the same amount of control IgG for ChIP-qPCR) overnight and then precipitated with Protein G Dynabeads (Cat#10004D, Thermo Scientific). Precipitates were washed sequentially using the following washes for 5 minutes each: low salt buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris-HCl pH 8.0, 500mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), 1X LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% deoxycholate, 1% NP-40) and twice in 1X TE buffer + 50 mM NaCl. Chromatin-antibody-beads were eluted in 50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS and de-cross-linked in 5 M NaCl solution at 65°C overnight. Sheared DNA was purified using the Zymo ChIP DNA Clean & Concentrator kit (Cat#D5201, Zymo) for ChIP–seq library preparation.
ChIP-seq libraries were prepared using the NEBNext Ultra DNA Library Prep kit for Illumina according to manufacturer’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
biological replicate_2
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| Data processing |
Demultiplexed fastq files were aligned to UCSC mm10 genome reference using bowtie2 (v2.26) with default arguments.
The generated BAM files were then analyzed using MACS2 (v2.1.0) with the following settings and the corresponding controls to identify significant binding peaks enriched in the target proteins: Tet1 ChIP-seq in WT and Tet1GT/GT C57BL/6 2iL-cultured ESCs: the MACS2 settings were “--nomodel --extsize 200 -q 0.01”; Oct4 ChIP-seq in WT and Tet1tm1Koh B6129S6F1 hybrid EpiLCs: the MACS2 settings were “-q 0.05”; Oct4 ChIP-seq in WT and Tet1GT/GT C57BL/6 EpiLCs: the MACS2 settings were “-q 0.05”.
The BAM files were first summarized and transformed into BigWig files with bamCompare command from deepTools2 (v3.3.1) using the following settings: --scaleFactorsMethod None --normalizeUsing RPKM --ignoreDuplicates
Genome_build: mm10
Supplementary_files_format_and_content: bigWig fiels provide peak tracks information
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| Submission date |
Feb 06, 2020 |
| Last update date |
Mar 02, 2020 |
| Contact name |
Kian Koh |
| E-mail(s) |
[email protected]
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| Organization name |
KULeuven
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| Department |
KU Leuven Department of Development and Regeneration
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| Lab |
Laboratory for Stem Cell and Developmental Epigenetics
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| Street address |
Herestraat 49, O&N IV - box 804
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| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE144868 |
Coordination of germ-layer lineage choice by TET1 during primed pluripotency (ChIP-seq) |
| GSE144869 |
Coordination of germ-layer lineage choice by TET1 during primed pluripotency |
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| Relations |
| BioSample |
SAMN14051436 |
| SRA |
SRX7687362 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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