H3K27ac_aNS_rep1
Mus musculus
Protocols: Primary V-SVZ-derived aNS cells were collected by centrifugation, the pellet was washed 2x in PBS and cross-linked with 1% PFA (in PBS, final conc.) for 23 min at 4°C, rotating. After quenching with glycine 0.1 M, the pellet was washed 2x in PBS+PI (protease inhibitor), shock-frozen in N2 and stored at -80 °C. 6-30 weeks-old C57Bl/6J mice were provided by the MPI for Brain Research Frankfurt, and the Central Animal Facility of the University Hospital Frankfurt. Adult neural stem-/progenitor cells were isolated from the lateral walls of the lateral ventricle and propagated at 37°C, 5% CO2 in the presence of EGF and FGF2 to allow for the formation of free-floating neurospheres. Adult neurospheres (aNS) were passaged every three to four days and 8 day old freshly passaged aNS cells were cross-linked for ChIP-seq. The pellet was thawed on ice and nuclei were lysed in L1 lysis buffer (50mM Tris-HCl pH 8.1, 2mM EDTA, 0.1% IGEPAL, 10% Glycerol, 1x PI). After centrifugation and removal of supernatant, the nuclear pellet was resuspended in SDS lysis buffer (50mM Tris-HCl pH 8.1, 10mM EDTA, 1% SDS, 1x PI), and sonicated for 7 cycles high power (30 s on and 90 s off) at 4 °C using a Bioruptor sonication device. Sheared chromatin was quantified using a Qubit 4.0 Fluorometer (dsDNA BR Assay kit) and pre-cleared with washed dynabeads for 1 h at 4°C, rotating. Immunoprecipitation was performed over night with 25 µg chromatin per IP and 2 µg of either α-Pbx1 rb (Cell Signalling, #4342), or α-H3K27ac rb (Diagenode, C15410196) antibodies. The next day, 50 µl dynabeads (1:1 mix protein A/G) per IP were added to each sample and incubated for 30 min at 4 °C, rotating. Beads were separated from solution with a magnet, the supernatant was discarded and beads were rinsed off the tube wall with low salt wash buffer (20mM Tris-HCl pH 8.1, 2mM EDTA, 1.0% IGEPAL, 150mM NaCl, 0.1% SDS, 1x PI). Subsequently, beads were washed as follows: 4x High salt wash buffer (20mM Tris-HCl pH 8.1, 2mM EDTA, 1.0% IGEPAL, 500mM NaCl, 0.1% SDS, 1x PI); 4x LiCl wash buffer (20mM Tris-HCl pH 8.1, 2mM EDTA, 1.0% IGEPAL, 500mM LiCl, 0.1% SDS, 1x PI); and 3x TE wash buffer (10mM Tris-HCl pH 8.1, 1mM EDTA, 1x PI). For elution, beads were incubated with 2% SDS in TE wash buffer (no PI), shaking, for 15 min at 25°C and 65°C, respectively, followed by decrosslinking of proteins over night at 65 °C, shaking. ChIP-enriched chromatin was purified on MicroChIP DiaPure columns (Diagenode) and successful ChIP was verified by qPCR. Library preparation was conducted with MicroPlex Library Preparation kit v2 (Diagenode).
ENA first public
1000-01-01
External Id
SAMEA114710136
INSDC center name
UOM-BCF
INSDC last update
2023-11-23T19:09:50Z
INSDC status
public
Submitter Id
E-MTAB-13565:H3K27ac_aNS_rep1
age
6 to 30
broker name
ArrayExpress
collection date
not collected
common name
house mouse
developmental stage
adult
genotype
wild type genotype
geographic location (country and/or sea)
not collected
individual
NB3
isolate
not applicable
organism part
brain
sample name
E-MTAB-13565:H3K27ac_aNS_rep1
scientific_name
Mus musculus
sex
mixed
strain
C57BL/6J