|
| Status |
Public on Apr 16, 2020 |
| Title |
180221_WT_6DL_UNC2400_S2 |
| Sample type |
SRA |
| |
|
| Source name |
wild-type Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129J
sequencing run#: 1
passage numbers: 68
treatment group: WT.6DL_UNC2400
sample preparation: ATAC-seq
|
| Treatment protocol |
ESC populations were untreated or treated throughout the 14 day differentiation treatment with the compounds indicated: A395, A395N, UNC1999, UNC2400, GSK343, GSKJ4, GSK5. The compound concentration was 1 micromolar for all compounds except GSK343 (3 micromolar)
|
| Growth protocol |
Murine embryonic stem cells were thawed (initial cell passage indicated), cultured in LIF containing media for 2 days (2DL), transferred to media containing 5 micromolar ATRA for a further 6 days (6DA) and transferred to LIF-containing media for a further 6 days (6DL). Where indicated, samples were sorted via FACS for GFP status (positive= pos or plus; negative = neg).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Frozen ESC pellets were thawed and assessed for viability followed by nuclei preparation and treatment with Tn5 transposase. The samples were subjected to PCR amplification and DNA purification. Adapted from Buenostro et al., 2015; doi:10.1002/0471142727.mb2129s109
Libraries were generated and optimized by PCR amplification, purified, and verified for proper size selection using a Agilent BioAnalyzer
To determine if the ATAC reactions were successful, an assessment of the overall enrichment of open chromatin regions was assayed by qPCR. Enrichment scores were calculated for each possible open vs closed region (2^(Ct_Open – Ct_Closed)). Scores over 10 were considered to be positively enriched.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
Illumina sequencing
|
| Data processing |
Libraries were sequenced with 75 bp single-end reads
Sequences were trimmed to 36 bp and mapped to mm9 using Bowtie 2 (v2.0.5) with default parameters (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
Any sample reads with less than 10M and/or peaks less than 20 M were excluded from further analysis. Poor quality reads (MAPQ<=30, reads mapping to chrM, chrY and patch chromosomes were also removed.
Reads were further filtered to remove PCR duplicates using Picard Tool MarkDuplicates (v2.6.0, https://broadinstitute.github.io/picard/). Accessible chromatin regions (peaks) were called using MACS2 v2.0.10. Default parameters were used except for the following: --keep-dup all -B -- nomodel --SPMR -q 0.05.
The signal intensity was calculated as the fold enrichment of the signal per million reads in a sample over a modelled local background using the bdgcmp function in MACS2. We then calculated the maximum signal intensity over every peak for conditions being compared using mapBed –o max and quantile normalized them across samples.
The average normalized signal intensity across replicates for each condition was then plotted in PCA. All downstream analyses (e.g. Spearman correlation coefficient analysis) were carried out using Bedtools v2.26.0 (https://bedtools.readthedocs.io/en/latest/) and R 3.4.1 (https://www.r-project.org/). Figures were plotted using ggplot2 (https://ggplot2.tidyverse.org/).
Genome_build: mm9
Supplementary_files_format_and_content: bedgraph
|
| |
|
| Submission date |
May 06, 2019 |
| Last update date |
Apr 16, 2020 |
| Contact name |
Lea Harrington |
| Organization name |
University of Montreal
|
| Department |
Department of Medicine
|
| Lab |
Institute for Research in Immunology and Cancer
|
| Street address |
2950 chemin de Polytechnique, Pavillon Marcelle-Coutu
|
| City |
Montreal |
| State/province |
Quebec |
| ZIP/Postal code |
H3T1J4 |
| Country |
Canada |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE130780 |
Telomere dysfunction cooperates with epigenetic alterations to impair murine embryonic stem cell fate commitment |
|
| Relations |
| BioSample |
SAMN11584829 |
| SRA |
SRX5799582 |
