|
| Status |
Public on Oct 24, 2018 |
| Title |
ATAC-seq_MEF_Ubc9-KO_rep3 |
| Sample type |
SRA |
| |
|
| Source name |
mouse embryonic fibroblast cells E13.5
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic fibroblast (MEF) cells
genotype/variation: Ubc9-KO
passages: 1
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-Seq: 10min formaldehyde fixation followed by sonication (histone marks) or Mnase digestion (transcription factors). Immunoprecipitation was performed using ChIP-IT kit (#53040, Active Motif) following manufacturer's protocol. Experiments were done in duplicates.
For ChIP-Seq: Libraries prepared using Microplex library Preparation kit v2 (C05010014, Diagenode) following manufacturer's protocol (V2 02.15). Libraries were size-selected (200-400bp) and clenaed-up using Agencourt AMPure XP beads (#A63881, Beckman).
For ATAC-seq: according to Buenrostro et al., Nature Methods, 2013. 30min treatment with Tn5 nuclease on isolated nuclei. Experiments were done in triplicates.
For ATAC-seq: Elute transposed DNA was PCR-amplified using barcode primers and purified using PCR Cleanup kit (QIAGEN)
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
For ChIP-seq single-end reads were mapped to the Mus musculus mm9 reference genome using bowtie1; unique matches with no more than 2 mismatches in the best alignment stratum were kept.
Following the ENCODE guidelines, mapped reads were deduplicated with the Picar toolkit v1.94, blacklisted regions removed with bedtools v2.19.1 and the degree of IP fragment clustering determined with phantompeakqualtools v2.0. Peak calling was performed with MACS2 with default parameters for regular peak calling and using the INPUT library for the estimation of the significance of peak enrichment. Consistency of peak calling among biological replicates and the final set of peaks per condition were determined using the Irreproducible Discovery Rate (IDR) approach.
For ATAC-seq paired-end reads were mapped using the end-to-end mode and the very-sensitive parametrization of bowtie2; concordant pairs were kept (including dovetail) with a maximum fragment size of 2 Kbp.
Mapped pairs were deduplicated with the Picar toolkit v1.94 and retained pairs were then classified according to their correspondent fragment; we restricted the analysis to fragment sizes sufficiently sampled in the ATAC-seq library by selecting read pairs coming from fragments between 50 bp and 2 Kbp.
Genome_build: mm9
Supplementary_files_format_and_content: tdf files were generated with igvtools v2.3.25. ChIP-seq and ATAC-seq libraries were merged by pooling the biological replicates. Prior to pooling, quality assurance clustering and principal component analysis were performed to identify and discard outlying replicates. The read coverage of the merged libraries was calculated over non-overlapping windows of 50 bp genome wide and library size normalization was applied to allow comparisons of the read coverage between samples: spike-in normalization for the ChIP-seq and quantile normalization for the ATAC-seq datasets.
Supplementary_files_format_and_content: bed files with selected peaks were generated by MACS2 and then filtered according to the IDR protocol; tab delimited format: chromosome start end MACS_peak_ID -log10(p-value)*10 . foldChange -log10(p-value) -log10(q-value) relativeSummitPosition
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| |
|
| Submission date |
Jun 15, 2018 |
| Last update date |
Oct 24, 2018 |
| Contact name |
Claudia Chica |
| Organization name |
Institut Pasteur
|
| Lab |
Biostatistics and Bioinformatics Hub
|
| Street address |
25-28 Rue du Dr Roux
|
| City |
Paris |
| ZIP/Postal code |
75015 |
| Country |
France |
| |
|
| Platform ID |
GPL21103 |
| Series (1) |
| GSE99009 |
SUMO safeguards somatic and pluripotent cell identities by enforcing distinct chromatin states |
|
| Relations |
| BioSample |
SAMN09428958 |
| SRA |
SRX4219806 |
