1000-01-01
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA114710140
External Id
UOM-BCF
INSDC center name
2023-11-23T19:09:50Z
INSDC last update
public
INSDC status
ERS17127297
SRA accession
E-MTAB-13565:PBX1_aNS_rep2
Submitter Id
6 to 30
week
age
ArrayExpress
broker name
not collected
collection date
house mouse
common name
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).
description
adult
developmental stage
wild type genotype
genotype
not collected
geographic location (country and/or sea)
NB2
individual
not applicable
isolate
Mus musculus
organism
Mus musculus
organism
brain
organism part
Mus musculus
scientific_name
mixed
sex
C57BL/6J
strain
PBX1_aNS_rep2
title