NextSeq 500 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
cells were extracted using TRIsure (Bioline, BIO-38033) following manufacturer' recommendations 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. Undifferentiated ST2 cells (day-1) were transfected with Lipofectamine RNAiMAX (Life Technologies, 13778150) according to manufacturer's instructions using 50 nM of gene-specific siRNAs against mouse Ahr (siAhr) (Dharmacon, M-044066-01-0005), Glis1 (siGlis1) (Dharmacon, M-065576-01-0005) or 50 nM of a negative control siRNA duplexes (siControl) (Dharmacon, D-001206-14-05). Cells were collected 48 h post-transfection. Cells were extracted using TRIsure (Bioline, BIO-38033) following manufacturer' recommendations TruSeq Stranded mRNA Library Prep kit (Illumina) was used to prepare the library for sequencing with 1 µg of RNA as starting material according to manufacturer's protocol. The library quality was checked using an Agilent 2100 Bioanalyzer and quantified using Qubit dsDNA HS assay Kit. The libraries were then adjusted to 4 nM
Experimental Factor: time
1
Experimental Factor: RNA interference
siRNA for AHR mRNA
Experimental Factor: protocol
RNA-Seq
Experimental Factor: immunoprecipitate
none