|
| Status |
Public on May 15, 2019 |
| Title |
4C_noteto_WT_chr1_reverseCTCF |
| Sample type |
SRA |
| |
|
| Source name |
4C_noteto_WT_chr1_reverseCTCF
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells (ESCs)
clone: 94.1 clone
|
| Treatment protocol |
no treatment
|
| Growth protocol |
Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle’s medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM β-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) in 8% CO2 at 37°C. After insertion of the rTetR-Dam vector (see below), cells were cultured in the presence of 250 µg/mL hygromycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The initial steps of 4C protocol were performed as published before (Splinter et al., 2012; Methods (PMID: 22929766)).
Briefly, cells are cross-linked using 2% formaldehyde for 10min at room temperature in PBS 10%FCS and nuclei are isolated, after which chromatin is digested with DpnII and subsequently ligated. After reversal of the cross-links, the DNA is purified and treated with the second restriction enzyme Csp6I. After a second re-ligation step, the sample is purified and a 4C PCR directly on this template for mouse genome viewpoints. In order to PCR amplify the ectopic TetO viewpoint, 4C templates were treated with Cas9 in vitro. Cas9 is targeted into the TetO repeats by using a sgRNA designed in between 4C viewpoint primers.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
S18_TetO_4C_Chris_ES_Wt_2_Chr1_VP22
|
| Data processing |
library strategy: 4C-seq
Single-end reads from each sample were mapped to the mouse genomes using the QuasR software (v 1.20.0, qAlign function, default settings).
4C signal quantification was done using qAlign (from QuasR, v 1.20.0) using as query a GRanges object containing the position of GATC sites. Library sizes are rescaled to the minimum library size
Running average of the signal was done using 21 steps
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files including genomic coordinates and enrichments
|
| |
|
| Submission date |
Mar 07, 2019 |
| Last update date |
Jun 01, 2024 |
| Contact name |
Luca Giorgetti |
| Organization name |
Friedrich Miescher Institute for Biomedical Research
|
| Street address |
Fabrikstrasse 24
|
| City |
Basel |
| ZIP/Postal code |
4056 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE128014 |
DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [4C] |
| GSE128017 |
DamC reveals principles of chromatin folding in vivo without crosslinking and ligation |
|
| Relations |
| BioSample |
SAMN11083073 |
| SRA |
SRX5493734 |
