GSM9001836: GFP exposed 3; Danio rerio; RNA-Seq
WIN 55,212-2 (WIN), bisphenol A (BPA), bisphenol S (BPS), and 3,3',5,5'-tetrabromobisphenol A (TBBPA) were dissolved in dimethyl sulfoxide (DMSO) in a stock solution of 10 mM. BPA, BPS and TBBPA were further diluted to 10 μM as an intermediate stock solution. Both the 10 mM and 10 μM solutions were stored at -20°C. Embryos were placed into 25 mL of E3 media in 50 mL tubes. Appropriate chemical volumes were added directly into the medium to get exposure concentrations of 120 or 200 nM for each bisphenol, and 108 or 180 nM for WIN. Additionally, suboptimal co-treatments of each bisphenol (120 nM) and WIN (108 nM) were used as well. Next, the embryos were placed back into the 28.5 °C incubator until time of fixation. Bisphenols A, S, and TBBPA were purchased from Sigma-Aldrich (St. Louis, MO, USA), and WIN was purchased from Cayman Chemicals (Ann Arbor, MI, USA). According to the literature, all chemical treatments are within normal exposure range. Embryos from the sp7-GFP transgenic line were exposed to 200 nM BPA for 24 hpf to 4 dpf (N = 400 embryos) and run against a control group at 4 dpf (N = 400 embryos). As EGPF takes longer than sp7 in situ to be detected in the opercle we slightly shifted our analysis time point and performed all transcriptomic analysis at 4 dpf. At 4d pf, the embryos were transferred 1.5 mL microcentrifuge tubes, and as much E3 was removed as possible without damaging the embryos. Before moving forward, the centrifuge was pre-cooled to 4°C (all centrifugation steps need to be performed at 4°C). Next, 150 μL of collagenase solution was added to dissociate the embryos in each tube, and they were placed an incubator at 30°C, with mild shaking, for 10 minutes. Following successful dissociation, a STOP solution (1.2 mL PBS w/ 10% FBS) was added to halt the dissociation step. Once cooled, set centrifuge to 700xg, spin for 5 minutes at 4°C, aspirate supernatant, and resuspend in 1 mL PBS with 2% FBS. Add 1 μL of DAPI. Next, Fluorescence-Activated Cell Sorting (FACS) was performed to sort all cells expressing sp7-GFP (only osteoblasts would express GFP at this stage), and the cells were sorted into 350 μL of RNA lysis buffer. RNA extraction was carried out to transport samples safely. The steps involved with dissociation, FACS, and RNA extraction (ZYMO Quick RNA MicroPrep Kit cat no. R1050) were performed by the Williamson, Clements Lab at St. Jude Children's Research Hospital. The RNA samples of the Control 4 dpf and the 200 nM BPA treatment were sent to the University of Mississippi Medical Center Molecular and Genomics Core Facility (www.umc.edu/genomicscore) for bulk quality control (QC) RNA-seq analysis. Reads were sequenced using the Illumina NextSeq 500 platform, and the Illumina BaseSpace Cloud Computing Program was used to assess the quality of sequenced reads.
GEO
GSM9001836_r1