Illumina HiSeq 4000 sequencing; Open chromatin profiling (ATAC-seq) of SGBS human preadipocytes (days 0, 2, 8 and 16 of differentiation to adipocytes) and iPSC-derived hypothalamic neurons (days 12, 16 and 27 of differentiation)
Open chromatin profiling (ATAC-seq) of SGBS human preadipocytes (days 0, 2, 8 and 16 of differentiation to adipocytes) and iPSC-derived hypothalamic neurons (days 12, 16 and 27 of differentiation)
SGBS were maintained and differentiated as previously described. Cells were grown in DMEM/F12 (1:1) media (Life Technologies #11330-032) with 10% fetal bovine serum, 5% penicillin-streptomycin solution (10,000 U/ml; gibco #15140122), 8mg/ml Panthotenic Acid, and 8mg/ml Biotin (Sigma; #B4639). Cells were allowed to grow to 70-80% confluency before splitting 1:3 with 0.25% Trypsin-EDTA(gibco; #25200056). To differentiate, cells were split into 6 well plates, allowed to reach 100% confluency over two days. After maintenance of confluency, day 0 cells were harvested, and the remaining cells were washed twice with 1x PBS and exposed to Quick-Diff media. Quick-Diff media consists of serum free DMEM/F12 media supplemented with 0.01mg/ml human transferrin (Sigma #T2252) , 20nM human insulin, 100nM cortisol, and 0.2nM Triiodothyronine. Day 2 cells were harvested two days after addition of quick-diff media. Cells were incubated for a total of 4 days in quick-diff media before 3FC media was added. The 3FC media consists of serum free DMEM/F12 media supplemented with 0.01mg/ml human transferrin (Sigma #T2252), 20nM human insulin, 100nM cortisol, 0.2nM Triiodothyronine, 25nM dexamethasone, 250uM 3-isobutyl-1-methylxanthine (IBMX), and 2uM rosiglitazone. Mature adipocytes (day 8 and 16) are maintained on 3FC media until collection.   Human induced pluripotent stem cells (hiPSCs) were differentiated into hypothalamic arcuate-like neurons, as previously described in PMID: 25555215. Briefly, the sonic hedgehog (SHH) signaling pathway was activated and transforming growth factor β (TGFβ) and bone morphogenetic protein (BMP) signaling were inhibited, followed by inhibition of NOTCH pathway, leading to neuronal induction, ventralization, and hypothalamic differentiation. After differentiation, brain-derived neurotrophic factor (R&D Bioscience # 248‐BD) was introduced to promote neuronal maturation of pro-opiomelanocortin (POMC) neurons. The neurons were then maintained in this medium supplemented with BDNF and B27 supplement until collection (Thermo Fisher #17504044).  We followed the Buenrostro paper ATAC seq protocol PMID: 25559105 for chromatin extraction ATAC-seq libraries for each cell type were generated according to the protocol outlined in Buenrostro et al. 2015 [25559105]. The cells were lysed, centrifuged, and frozen at -80C until all time points were collected. Final processing of all pellets was performed together. Transposed DNA fragments were PCR amplified using 5- 7 PCR cycles. PCR cycle number was determined using qPCR reactions where the additional cycle numbers were those that corresponded to the inflection point of the qPCR curve.