|
| Status |
Public on Jan 01, 2015 |
| Title |
ESC_FAIRE-Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
FAIRE-seq of mESC chromatin
|
| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cells
|
| Treatment protocol |
n/a
|
| Growth protocol |
E14 Mouse embryonic stem cells (ES-E14Tg2a, ATCC #CRL-1821) were maintained on gelatin-coated tissue culture plates in N2/B27 medium (DMEMF12 (Gibco) and Neurobasal (Gibco) in a 1:1 ratio supplemented with N2 (home-made or StemCells), B27 (Invitrogen), L-glutamine and β-mercaptoethanol) containing 1uM MEK inhibitor PD032590 and 3 uM GSK3 inhibitor CH99021 (Axon Medchem BV, The Netherlands) And 10^3 U/ml LIF (Millipore). E3 Epiblast Stem cells were a gift from Boris Greber (Max Planck Institute, Muenster, Germany) and were expanded on a feeder layer of mitomycin C-treated MEFs in MEF-conditioned medium supplemented with 5ng/ml FGF2 (Peprotech). Two days prior to formaldehyde crosslinking, the expanded EpiSC cultures were enzymatically isolated from the MEF feeder layer using Dispase digestion (1mg/ml, 15 minutes) and transferred onto 15 cm fibronectin-coated plates for further feeder-free culture in MEF-conditioned medium supplemented with FGF-2. Neural Stem cells were maintained in N2B27 medium supplemented with 10ng/ml each of FGF-2 and EGF (Peprotech). Mouse Embryo Fibroblasts were derived from E16.5 embryos and maintained in DMEM and 10% FBS.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
FAIRE was performed as described (Giresi et al. 2007; Giresi and Lieb 2009) DNA fragments are prepped for sequencing using the recommended protocol except that samples are amplified prior to gel extraction. Adaptor-ligated DNA fragments amplified using PCR and ranging from 150 to 400 bp were subsequently purified from agarose gels (Qiagen Gel Extraction kit).
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~150-400 bp were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
FAIRE-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Data processing |
Sequencing was performed using the Illumina GAIIx genome analyzer and FASTQ files were generated using CASAVA 1.8
FAIRE-seq and control genomic DNA reads were aligned to the mouse genome (mm9) using ELAND
Peak calling was performed with Qeseq using default parameters
Genome_build: mm9
Supplementary_files_format_and_content: processed data presented as bed files containing the locations of all peaks as well as wig files showing peak location and intensity.
|
| |
|
| Submission date |
Jun 16, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Matthew Murtha |
| E-mail(s) |
matthew.murtha@nyumc.org
|
| Phone |
2129929171
|
| Organization name |
NYU School of Medicine
|
| Department |
Microbiology
|
| Lab |
Dailey
|
| Street address |
550 First Ave
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10016 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE58520 |
Comparative FAIRE-seq analysis reveals distinguishing features of the chromatin structure of ground state- and primed pluripotent cells |
|
| Relations |
| BioSample |
SAMN02863159 |
| SRA |
SRX603327 |
| Named Annotation |
GSM1412927_E14-1.qeseq.enr.wig.gz |
