 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Mar 02, 2020 |
| Title |
ATACseq_Rmt_EpiLC_GTB6_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Tet1 gene-trap (GT) EpiLC rescued with catalytic mutant Tet1(Rmt)
|
| Organism |
Mus musculus |
| Characteristics |
cell type: EpiLC
genotype: Rescued_with_catalytic_mutant
strain: C57BL/6
|
| 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 ug/10cm2 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).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
50,000 freshly isolated cells were washed once with 100 ul PBS and resuspended in ice-cold 50 ul lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin) for 3 min. The suspension of nuclei was then centrifuged for 10 min at 500 g at 4°C, followed by one wash with lysis buffer without Tween-20 and Digitonin. The visible pellets were incubated with 50 ul transposition reaction mix (25 l 2x TD buffer, 2.5 ul (or 5 ul) Tn5 Transposase, 16.5 ul PBS, 0.5 l 1% Digitonin, 0.5 ul 10% Tween-20 and nuclease-free H2O) (Cat#FC-121-1030, Illumina) at 37°C for 30 min with 500×g mixing. DNA was isolated using Zymo DNA clean and concentrator-5 kit (Cat#D4014, Zymo).
Library amplification was done by two sequential PCR reactions (5 and 8 cycles, respectively) using NEBNext High-Fidelity 2X PCR Master Mix (Cat#M0541S, New England BioLabs).
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
biological replicate_2
|
| Data processing |
Demultiplexed fastq files were analyzed using automated ENCODE ATAC-seq pipeline (http://doi.org/10.5281/zenodo.156534) developed by Anshul Kundaje’s laboratory with default arguments.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig fiels provide peak tracks information
|
| |
|
| Submission date |
Feb 06, 2020 |
| Last update date |
Mar 02, 2020 |
| Contact name |
Kian Koh |
| E-mail(s) |
[email protected]
|
| Organization name |
KULeuven
|
| Department |
KU Leuven Department of Development and Regeneration
|
| Lab |
Laboratory for Stem Cell and Developmental Epigenetics
|
| Street address |
Herestraat 49, O&N IV - box 804
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE144867 |
Coordination of germ-layer lineage choice by TET1 during primed pluripotency (ATAC-seq) |
| GSE144869 |
Coordination of germ-layer lineage choice by TET1 during primed pluripotency |
|
| Relations |
| BioSample |
SAMN14051410 |
| SRA |
SRX7687350 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
