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Sample GSM2356842 Query DataSets for GSM2356842
Status Public on Oct 04, 2017
Title input DNA
Sample type SRA
Source name neuronal stem cells
Organism Mus musculus
Characteristics strain: C57BL/6J
genotype: wildtype
age: E12.5 embryo
fixation: 20' 1.5 mM EGS, 10' 1% FA
antibody: none
Treatment protocol none
Growth protocol Embryonic NSCs from E12.5 embryos were cultured under proliferation conditions in Neurobasal-A medium supplemented with GlutaMAX (1%), glutamine (0.5%), Penicillin/Streptomycin (1%), rhFGF-2 (20 ng/ml), B-27 minus Vitamin A (2%) (all from Invitrogen) and rhEGF (20 ng/ml, Sigma-Aldrich).
Extracted molecule genomic DNA
Extraction protocol Embryonic NSCs (approx. 10 x 10^6 cells) were fixed with 1.5 mM ethylene glycol bis[succinimidyl succinate] in DMSO for 20 min at RT, followed by adding formaldehyde to a final concentration of 1%, incubated for 10 min at RT and finally quenched by 0.2 M glycine. Cells were then lysed and sonicated with a microtip for 10 seconds followed by a 1 min break for 10 cycles. After adding 1% Triton-X to the sonicated lysates, ChIP was performed overnight at 4°C with Dynabeads M-280 Sheep anti-Rabbit IgG (Thermo Fisher, 11203D) linked to rabbit anti-E2A (1.5 µg, sc-349X, Santa Cruz Biotechnology). 10% of sonicated suspension was kept as input control. After washing and elution of chromatin-antibody complexes from the beads at 65°C overnight, crosslinking was reversed by proteinase K and RNase digestion. Genomic DNA and DNA from the sonicated input were purified using a PCR Purification Kit (Qiagen).
Library preperation was performed from 2 ng of ChIP DNA using the Kapa Hyper Prep Kit with PCR library amplification (#KK8504, KapaBiosystems) according to the manufactur's protocol. Shortly, ChIP DNA was end-repaired and A-tailed before adapter ligation. After adapter ligation, the ChIP DNA was purified with Agencourt AMPure XP beads (#A63880, Beckman Coulter) and size-selected for 360-600 bp using the Pippin Prep System from Saga Science. The size-selected library is amplified by PCR using the Kapa Hyper Prep Kit (#KK8504,KapaBiosystems) and purified terminally with Agencourt AMPure XP beads (#A63880, Beckman Coulter).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 3000
Data processing Reads were aligned to the mouse mm9 reference genome using BWA-MEM version 0.7.13 with default parameter settings.
PCR duplicates were removed using Picard Tools version 1.119.
Peaks were called using MACS2 version 2.1.0 with a FDR threshold of 0.01.
Genome_build: mm9
Supplementary_files_format_and_content: The .bed files containing the peak positions were generated with MACS2 version 2.1.0.
Submission date Oct 20, 2016
Last update date May 15, 2019
Contact name Franziska Greulich
Organization name TU München
Department Metabolic Programming
Lab AG Uhlenhaut
Street address Gregor-Mendel-Str. 2
City Freising
ZIP/Postal code 85354
Country Germany
Platform ID GPL21493
Series (1)
GSE88991 Genome-wide E2A binding map in murine neuronal stem cells
BioSample SAMN05930746
SRA SRX2254826

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data not applicable for this record
Processed data provided as supplementary file

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