|
| Status |
Public on Apr 11, 2012 |
| Title |
female-mESCs-PGK12.1-replicate-2 |
| Sample type |
SRA |
| |
|
| Source name |
Undifferentiated mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Undifferentiated mouse embryonic stem cells
sex chromosome ploidy: XX
|
| Growth protocol |
Feeder dependent mESCs (TT2, TT2-G9a 22.10 and XTX) were grown on a mono-layer of mitomycin C treated MEFs and feeder independent mESCs (E14, PGK12.1, EED-l7Rn5-3354SB/l7Rn5-3354SB ,) were grown on gelatin-coated flasks. Culture media consisted in DMEM (Gibco) except for E14 that were grown in Glasgow medium supplemented with 2mM L-Glutamine, 0.1mM NEAA and 1mM sodium pyruvate (Gibco). All mESC media contained 15% FBS (Gibco), 10-4 M b-mercaptoethanol (Sigma), 1000 U/ml of leukaemia inhibitory factor (LIF, Chemicon) and cells were cultivated under 8% CO2. TT2 and TT2-G9a 22.10. Female heterozygous DXTX cells were subcloned to isolate an XO line containing only the deleted X chromosome. NPCs were derived in N2B27 (StemCell Sciences) and cultured with N2B27 + 10ng/mL EGF and FGF (Preprotech) on gelatin-coated flasks. Primary MEFs were obtained from E13.5 embryo dissociation (C57BL/6 background) and passaged three times in DMEM containing 10% FBS (Gibco) and 10-4 M b-mercaptoethanol (Sigma).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
3C DNA was hybridised with a pool of 5C oligonucleotide designed upstream (FORWARD) or downstream (REVERSE) HindIII restriction sites. Ligation of FORWARD and REVERSE oligonucleotides was then performed to generate a 5C library, which was amplified using T3 and T7 primers that hybridise to the respective tails of all 5C-FORWARD and 5C-REVERSE oligonucleotides. 5C libraries were then prepared for paired-end sequencing on a Genome Analyzer II machine according to Illumina's instructions (Kit PE-102-1001).
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
ligated DNA oligonuclotides
|
| Data processing |
mapping: each side of the paired end read, which were independently mapped to a pseudo-genome of all possible 5C oligonucleotides sequences using the novoalign mapping algorithm (V2.05 http://novocraft.com) with default settings. After mapping, if both of the paired end reads could be uniquely mapped to a 5C primer, a 5C interaction was assembled. Invalid interactions between the same primer or between primers of the same type were removed as these would represent a mapping artifact or an issue with the 5C technique.
Some 5C oligonucleotides were found to show artifactual ligation patterns (e.g. constitutively high signal or no signal at all) and were therefore flagged as outliers and removed from the analysis. As oligonucleotide was flagged as outlier if its median interaction count was 2.5 times higher than the interquartile range of the median counts of the whole oligonucleotide set in at least half of the experiments. Median counts were computed using primers that had non-null count values.
Genome Build:
female-mESCs-PGK12.1-replicate-2.matrix: Mapping was done on a virtual genome made of all possible 5C oligonucleotide ligation products. Coordinates of FORWARD and REVERSE oligonucleotides are based on mm9 assembly
|
| |
|
| Submission date |
Feb 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Elphege Pierre Nora |
| E-mail(s) |
elphege.nora@curie.fr, elphnora@gmail.com
|
| Organization name |
Institut Curie
|
| Department |
Mammalian developmental epigenetics
|
| Lab |
Edith Heard
|
| Street address |
11-13 rue Pierre et Marie Curie
|
| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE35721 |
Spatial organisation of the X inactivation center |
|
| Relations |
| SRA |
SRX119476 |
| BioSample |
SAMN00788647 |
