Illumina HiSeq 2000 sequencing; Temporal enhancer profiling of parallel lineages identifies AHR and GLIS1 as regulators of mesenchymal multipotency
Temporal enhancer profiling of parallel lineages identifies AHR and GLIS1 as regulators of mesenchymal multipotency
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
Experimental Factor: time
9
Experimental Factor: RNA interference
none
Experimental Factor: protocol
ChIP-Seq
Experimental Factor: immunoprecipitate
anti-H3K27ac