Mus musculus
Protocols: Forelimbs buds were manually dissected from ca. 250 RjOrl:SWISS outbred 10.5 dpc mouse embryos. Chromatin immunoprecipitation was performed as in Cantù et al., 2013. Briefly, the tissue was dissociated to a single cell suspension with collagenase (1 g/l in PBS) for 1 hour at 37° C, washed and cross-linked in 20 ml PBS for 40 min with the addition of 1.5 mM ethylene glycol-bis(succinimidyl succinate) (Thermo Scientific, Wal- tham, MA, USA), for protein-protein cross-linking (Schuijers et al., 2014.), and 1% formaldehyde for the last 20 min of incubation, to preserve DNA-protein interactions. The reaction was blocked with glycine and the cells were subsequently lysed in 1 ml hepes buffer (0.3% SDS, 1% Triton-X 100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES). Chromatin was sheared using Covaris S2 (Covaris, Woburn, MA, USA) for 8 min with the following set up: duty cycle: max, intensity: max, cycles/burst: max, mode: Power Tracking. The sonicated chromatin was diluted to 0.15% SDS and incubated overnight at 4°C with 1 g of anti-Bcl9 (Abcam, ab37305) or IgG and 50 l of protein A/G magnetic beads (Upstate). The beads were washed at 4°C with wash buffer 1 (0.1% SDS, 0.1% deoxycholate, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), wash buffer 2 (0.1% SDS, 0.1% sodium deoxycho- late, 1% Triton X-100, 0.5 M NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), wash buffer 3 (0.25 M LiCl, 0.5% sodium deoxycholate, 0.5% NP-40, 1 mM EDTA, 0.5 mM EGTA, 20 mM HEPES), and finally twice with Tris EDTA buffer. The chromatin was eluted with 1% SDS, 0.1 M NaHCO3, de-crosslinked by incubation at 65°C for 5 h with 200 mM NaCl, extracted with phenol-chloroform, and ethanol precipitated. The immunoprecipitated DNA was used as input material for DNA deep sequencing. We used commands in HOMER to create a bedGraph formatted files that can then be uploaded as a custom track to the genome browser. We used the following command from the HOMER package: makeUCSCfile <tag directory> -o auto. These files can be visualised in UCSC genome browser and IGV. Libraries were prepared starting with 5 ng of ChIP-enriched DNA as starting material and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Library molarity and quality was assessed with the Qubit and Tapestation using a DNA High sensitivity chip (Agilent Technologies).
ENA-FIRST-PUBLIC
2019-02-21T17:03:56Z
ENA-LAST-UPDATE
2019-02-11T12:44:49Z
External Id
SAMEA5331389
INSDC center name
Wallenberg Centre for Molecular Medicine (WCMM), Linkoping University. Department of Clinical and Experimental Medicine (IKE), Faculty of Health Sciences, Linkoping University, Linkoping, Sweden
INSDC first public
2019-02-21T17:03:56Z
INSDC last update
2019-02-11T12:44:49Z
INSDC status
public
Submitter Id
E-MTAB-7652:Sample 4
age
embryo
broker name
ArrayExpress
common name
house mouse
developmental stage
embryonic day 10.5
genotype
wild type genotype
individual
pooled material
organism part
forelimb
sample name
E-MTAB-7652:Sample 4
scientific_name
Mus musculus
strain
Swiss