Protocols: 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.
ENA first public
2021-05-22
ENA last update
2021-05-22
External Id
SAMEA8742859
INSDC center alias
UNIVERSITY OF CHICAGO
INSDC center name
UNIVERSITY OF CHICAGO
INSDC first public
2021-05-22T00:23:43Z
INSDC last update
2021-05-22T00:23:43Z
INSDC status
public
Submitter Id
E-MTAB-10462:SGBS day0 rep1
ancestry
caucasian
biological replicate
1
broker name
ArrayExpress
cell line
SGBS
cell type
preadipocyte
common name
human
day of differentiation
0
developmental stage
embryo stage
disease
Simpson-Golabi-Behmel syndrome
genotype
wild type genotype
organism part
adipose tissue
sample name
E-MTAB-10462:SGBS day0 rep1
technical replicate number
1