Illumina HiSeq 2000 sequencing; Temporal enhancer profiling of parallel lineages identifies AHR and GLIS1 as regulators of mesenchymal multipotency
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Bone
Cell type
Osteoblasts
NA
NA
Attributes by original data submitter
Sample
ENA first public
2019-02-08
ENA last update
2018-05-30
ENA-CHECKLIST
ERC000011
External Id
SAMEA4699861
INSDC center alias
Life Sciences Research Unit, University of Luxembourg
INSDC center name
Life Sciences Research Unit, University of Luxembourg
INSDC first public
2019-02-08T17:02:37Z
INSDC last update
2018-05-30T17:06:56Z
INSDC status
public
Submitter Id
E-MTAB-6840:Ob-Day3-H3K27
broker name
ArrayExpress
cell line
ST2
cell type
osteoblast
common name
house mouse
organism part
bone marrow
progenitor cell type
stromal cell
sample name
E-MTAB-6840:Ob-Day3-H3K27
Sequenced DNA Library
library_name
Ob-Day3-H3K27_s
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
chip-seq: cells were lysed in 1.7 mL of ice-cold lysis buffer [5 mM 1,4-Piperazinediethanesulfonic acid (PIPES) pH 8.0 (Carl Roth, 9156.3); 85 mM potassium chloride (KCl) (PanReac AppliChem, A2939); 0.5 % 4-Nonylphenyl-polyethylene glycol (NP-40) (Fluka Biochemika, 74385)] containing PI and incubated for 30 minutes on ice The mouse bone marrow stromal cell line ST2 established from Whitlock-Witte type long-term bone marrow culture of BC8 mice (Ogawa et al., 1988) was used during all experiments. Cells were grown in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Life Technologies, 32404014) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, 10270-106, lot #41F8430K) and 1% L-Glutamine (Lonza, BE17-605E) in a constant atmosphere of 37°C and 5 % CO2. For differentiation into adipocytes and osteoblasts, ST2 cells were seeded 4 days before differentiation (day-4), reached 100% confluency after 48 hours (day-2) and were further maintained for 48 hours post-confluency (day 0). Adipogenic differentiation was subsequently initiated on day 0 (D0) by adding differentiation medium I consisting of growth medium, 0.5 mM isobutylmethylxanthine (IBMX) (Sigma-Aldrich, I5879), 0.25 µM dexamethasone (DEXA) (Sigma-Aldrich, D4902) and 5 µg/mL insulin (Sigma-Aldrich, I9278). From day 2 (D2) on differentiation medium II consisting of growth medium, 500 nM rosiglitazone (RGZ) (Sigma-Aldrich, R2408) and 5 µg/mL insulin (Sigma-Aldrich, I9278) was added and replaced every 2 days until 15 days of differentiation. Osteoblastic differentiation was induced with growth medium supplemented with 100 ng/mL bone morphogenetic protein-4 (BMP-4) (PeproTech, 315-27). Same media was replaced every 2 days until 15 days of osteoblastogenesis. chip-seq: Chromatin immunoprecipitation of histone modifications was performed on indicated time points of adipocyte and osteoblast differentiation. Cells were grown on 10 cm2 dishes. First, chromatin was cross-linked with formaldehyde (Sigma-Aldrich, F8775-25ML) at a final concentration of 1% in the culture media for 8 minutes at room temperature. Then, the cross-linked reaction was quenched with glycine (Carl Roth, 3908.3) at a final concentration of 125 mM for 5 minutes at room temperature. The formaldehyde-glycine solution was removed and cells were washed twice with ice-cold phosphate-buffered saline (PBS) (Lonza, BE17-516F) containing cOmpleteTM mini Protease Inhibitor (PI) Cocktail (Roche, 11846145001). Then, cells were lysed in 1.7 mL of ice-cold lysis buffer [5 mM 1,4-Piperazinediethanesulfonic acid (PIPES) pH 8.0 (Carl Roth, 9156.3); 85 mM potassium chloride (KCl) (PanReac AppliChem, A2939); 0.5 % 4-Nonylphenyl-polyethylene glycol (NP-40) (Fluka Biochemika, 74385)] containing PI and incubated for 30 minutes on ice. The cell lysates were then centrifuged at 660 xg for 10 min at 7°C and the pellet was resuspended in 400 μL of ice-cold shearing buffer [50 mM Tris Base pH 8.1 (Carl Roth, 4855.2); 10 mM Ethylenediamine tetraacetic acid (EDTA) (Carl Roth, CN06.3); 0.1 % Sodium Dodecylsulfate (SDS) (PanReac Applichem, A7249); 0.5 % Sodium deoxycholate (Fluka Biochemika, 30970)] containing PI. Chromatin was sheared with a sonicator (Bioruptor®Standard Diagenode, UCD-200TM-EX) during 20 cycles at high intensity (30 s off and 30 s on) for the ST2 cells differentiated into adipocytes and osteoblasts and 25 cycles at high intensity (30 s off and 30 s on) for the ST2 differentiated into osteoblasts for 9 days on. The sheared cell lysate was then centrifuged at 20817 xg for 10 minutes at 7°C and the supernatant containing the sheared chromatin was transferred to a new tube. Antibodies: H3K27ac (from Abcam, item #ab4729), H3K36me3 (Abcam, ab9050), H3K4me3 (Millipore, 17-614). TruSeq unstranded mRNA Library Prep kit (Illumina) was used to prepare libraries