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Supplementary Materials and Methods Expression analysis of ADCYAP1R1 in transfected HEK293T cells HEK293T cells were plated at approximately 5 X 105 cells in 2, 6-well culture plates with phenol red free DMEM (cat # 12-917F; Lonza BioWhittaker, Walkersville, MD) supplemented with L-glutamine (4mM; cat # 25030-081; ThermoFisher Scientific) and charcoal-stripped FBS (10%; cat # S11695H; Atlanta Biologicals, Inc., Flowery Branch, GA). 25 hours later, the cells were transfected with either 2g of a plasmid vector containing green fluorescent protein (GFP) or 3g of a plasmid containing the full-length human estrogen receptor, pCMVhERkindly provided by Dr. Ann Nardulli) in 2mls of Opti-MEM (cat # 31985-070; ThermoFisher Scientific) plus 10l of Lipofectamine 2000 (cat # 11668; ThermoFisher Scientific). The cells were transfected for 6.5 hours after which the cell culture media was replaced with hormone free DMEM (as above) plus penicillin/streptomycin (1%; cat # 11668, ThermoFisher Scientific). After 21 hours, the cells from each transfection were combined and re-plated in hormone free media across 12 wells (24 total). After 24 more hours, 6-wells of cells for each transfection were grown in media with either 10nM 17-Estradiol (E2) (cat # E2758; Sigma-Aldrich, St. Louis, MO) or EtOH only for 16.5 hours. mRNA was extracted from the cells using the RNeasy mini kit (cat # 74104; Qiagen, Inc., Valencia, CA). mRNA was converted to cDNA using the RT2 First Strand Kit (cat # 330401, Qiagen, Inc.). Gene expression of ADCYAP1R1 was assayed using TaqMan Gene Expression Assays: Hs01027974_m1 (ADCYAP1R1) and Hs02758991_g1 (GAPDH, endogenous control) with TaqMan Gene Expression Master Mix (cat # 4369016) (ThermoFisher Scientific). The 2-∆∆Ct method was used to compare foldchange in expression between each condition and treatment group. Successful transfection of hER, which is not endogenously expressed in HEK293T cells, was confirmed using a TaqMan expression assay (ESR1, Hs00174860_m1, ThermoFisher Scientific). Additionally we observed 70-90% transfection efficiency for GFP by assessing florescence under a compound microscope. Cross-linking chromatin immunoprecipitation (X-ChIP) HEK293T cells were transfected with 3g of pCMV-hER (as described above) and treated with 10nM E2 for 2 hours. Prior to treatment with E2, cells were grown in hormone-free media: DMEM with phenol red (cat # 12-741F, Lonza BioWhittaker, Walkersville, MD) supplemented with additional L-glutamine (1%, cat # 25030-081; ThermoFisher Scientific) and charcoal stripped FBS (10%, cat # 12676011; ThermoFisher Scientific) for 41.5 hours. Following treatment with E2, the cells from each of 6-wells were gently pelleted and rinsed with 1 x phosphate buffered saline (PBS). After removing the PBS, 940ul of 1% formaldehyde in 1 x PBS (made from 37%; cat # F8775, Sigma Aldrich, St. Louis, MO) was added to the cells and placed at 37 degrees Celsius for 7 minutes. Glycine was then added at a final concentration of 125mM and incubated for 5 minutes. From this point forward all samples were kept on ice or at 40 Celsius (C). Cells in lysis buffer were sonicated using the Bioruptor (Diagenode Inc., Denville, NJ) for 30 cycles of 30 seconds on/ 30 seconds off. 10% of the total sonicated product was placed at -800 C to be used later as non-immunoprecipitated “input” DNA. For each of 6 replicates, 20g of sheared, DNA-protein complex (in Chip dilution buffer) was immunoprecipitated using 2µg of a mouse monoclonal antibody to ER (D-12) X (cat # sc-8005 X; Santa Cruz, Dallas, TX). DNA-protein complexes bound to the antibody were incubated for 4.5 hours with a 50/50 mixture of magnetic beads covalently coupled with recombinant Protein A and Protein G (Dynabeads Protein A, cat # 10002D; Dynabeads Protein G, cat # 10004D, ThermoFisher Scientific) blocked with bovine serum albumin (BSA) and yeast RNA (cat # AM7118, ThermoFisher Scientific). Bound beads were rinsed with several buffers in the following order: low salt buffer, high salt buffer, lithium chloride buffer and Tris-EDTA (TE) buffer. The contents of each buffer are provided below. Remaining, bound DNA/protein complexes were eluted from the beads by adding elution buffer and heating the sample at 550 C for 15 minutes. To reverse the crosslinking we added RNase A (50g, cat # 10109169001, Roche, Indianapolis, IN) for 2 hours at 370 C and then Proteinase K (approx. 35g, cat # 03115828001, Roche, Indianapolis, IN) overnight (12+ hours) at 650 C. The DNA was purified using a standard phenol chloroform extraction and ethanol precipitation with glycogen (40g; cat # 77534, Affymetrix, Santa Clara, CA). Quantitative PCR was performed using SYBR green master mix (cat # 330523, Qiagen Inc., Valencia, CA). Primers used for this experiment are listed in Supplementary Table 1. Percent input was calculated as described in the following document: http://www.thermofisher.com/us/en/home/life-science/epigenetics-noncodingrna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chipanalysis.html ChIP solutions: SDS Lysis Buffer (10mls) 50mM Tris HCl -- from 1M stock = 500ul 10mM EDTA -- from 0.5M stock = 200ul 1% SDS --- from 20% stock = 500ul 1mM PMSF – from 100mM stock isopropranol = 100ul Bring up to volume in RNase-free water + 1 Roche Proteinase Inhibitor (PI) Complete tablet ChIP Dilution Buffer (10mls) 16.7mM Tris-HCl -- from 1M stock = 167ul 1.2mM EDTA – from 0.5M stock = 24ul 167mM NaCl – from 5M stock =334ul 1.1% TritonX – from 100% = 110ul 0.01% SDS – from 20% stock = 5ul Bring up to volume in RNase-free water +1 Roche PI Complete tablet High-Salt Buffer (10mls) 20mM Tris-HCl – from 1M stock = 200ul 2mM EDTA – from 0.5M stock = 40ul 1% Triton X = 100ul 0.1% SDS – from 20% stock = 50ul 500mM NaCl – from 5M stock = 1ml Bring up to volume in RNase-free water +1 Roche PI Complete tablet Low-Salt Buffer (10mls) 20mM Tris-HCl – from 1M stock =200ul 2mM EDTA – from 0.5M stock =40ul 1% TritonX = 100ul 0.1% SDS – from 20% stock = 50ul 150mM NaCl – from 5M stock =300ul Bring up to volume in RNase-free water +1 Roche PI Complete tablet LiCl Buffer (10mls) 10mM Tris HCl –from 1M stock = 100ul 1mM EDTA – from 0.5M stock = 20ul 1% Sodium Deoxycholate –from 10% stock = 1ml 250mM LiCl – from 1M stock =2.5ml 1% NP40 – from 10% stock = 1ml Bring up to volume in RNase free water +1 Roche PI Complete tablet TE buffer (10mls) 10mM Tris HCl – from 1M stock = 100ul 1mM EDTA –from 0.5M stock = 20ul Bring up to volume in RNase free water +1 Roche PI Complete tablet Elution Buffer (10mls) 50mM Tris HCl – from 1M stock = 500ul 10mM EDTA – from 0.5M stock =200ul 1% SDS – from 20% stock =500ul Bring up to volume in RNase free water (no PI—keep at room temp)
