|
| Status |
Public on Aug 06, 2020 |
| Title |
E14_FCS_226_Mtf2_A.mapUnique |
| Sample type |
SRA |
| |
|
| Source name |
embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14
genotype: WT
treatment: EED 226
chip antibody: Aviva System Biology ARP34292, lot QC49692-42166
|
| Treatment protocol |
Where indicated ESCs were treated with 10 µM EED226 for 4 days to inhibit EED function. Full knockout of Ring1b was induced through treatment with Tamoxifen (OHT) for 2 days. For H2AK119u depletion cells were incubated in medium containing 5uM of MG132 for 6 hours
|
| Growth protocol |
mESC were grown in serum+LIF medium as described in Marks, H. et al. (doi:10.1016/j.cell.2012.03.026)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin extraction and ChIP were performed as described in Kloet, S. L. et al. (doi:10.1038/nsmb.3248 )
Five ng/sample of ChIP were prepared for sequencing with the Kapa Hyper-prep Kit (Kapa Biosystems) using NEXTflex adapters (Bio Scientific) and amplified with ten cycles of PCR. Libraries were size-selected to obtain 300bp fragments using E-gel (Invitrogen) and sequenced on an Illumina NextSeq 500 machine to obtain 75bp reads
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
ChIP for Mtf2 in E14 cells treated with Eed226
processed data file:
E14_FCS_226_Mtf2.mapUnique.bw
|
| Data processing |
Base-calling was performed by the Illumina CASAVA software.
Reads from this study and from Perino et al, Nat. Gen (2018) were trimmed to 42bp with fastx_trimmer (version 0.0.13.2), and in case of paired-end sequencing only read_1 was used for analysis to ensure maximum comparability
All fastq files were mapped using bwa (version 0.7.10-r789), filtered to retain only uniquely mapping reads using mapping quality of 30 and samtools (version 1.7, flag -F 1024)
Peaks were called with MACS2-2.7 with qvalue 0.0001 using --call-summits for transcription factors and --broad for H3K27me3. Only peaks independently called in both replicates were used for downstream analysis
High-confidence peaks for each mark were obtained by merging peaks called in both replicates and overlapping by at least 50% of their length, and combined to obtain the list of all PRC2 peaks.
Coverage files in bigWig format were created using bam2bw (https://bitbucket.org/simonvh/bam2bw/) with the -e auto and -c parameters
Clustering was performed with fluff v3.0.2 https://fluff.readthedocs.io/en/latest/ using the -g option to detect dynamics
Samples supplemented with Drosophila chromatin were mapped on a combined mm10-dm6 genome, filtered to remove multimappers and duplicates. The resulting bam files were split according to the species of origin and dm6 reads were used to calculate a per-sample scaling factor relative to the less deeply sequenced sample. ChIPseq were first normalized based on the number of spike-in reads to obtain the number of reads per million of spike-in reads, and further normalized to account for the potentially varying starting amount of spike-in in different samples.
Genome_build: mm10
Supplementary_files_format_and_content: Coverage files in bigWig format, high-confidence PRC2 peaks and clustered peaks, normalized bam files for spike-in ChIP
|
| |
|
| Submission date |
Jun 20, 2019 |
| Last update date |
Aug 06, 2020 |
| Contact name |
Gert Jan Veenstra |
| E-mail(s) |
g.veenstra@science.ru.nl
|
| Phone |
+31 24 3610541
|
| Organization name |
Radboud University
|
| Department |
Molecular Developmental Biology
|
| Street address |
Geert Grooteplein 28
|
| City |
Nijmegen |
| State/province |
Not US or Canada |
| ZIP/Postal code |
6525 GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL19057 |
| Series (1) |
| GSE133085 |
Two distinct functional axes of positive feedback-enforced PRC2 recruitment in mouse embryonic stem cells |
|
| Relations |
| BioSample |
SAMN12099423 |
| SRA |
SRX6098698 |
