title: GSM8241840: E125_PGC_Male_ChIP_TRIM28_1; Mus musculus; ChIP-Seq
library_construction_protocol: Gonads from time-mated CD1 mice were dissected at E12.5. Following dissection, gonads were seperated from the mesonephrous and digested with 0.05% trypsin. Digestion was stopped with 10% FBS + High Glucose DMEM with 1x DNAse. Cells were resuspended in 10% FBS + Hgih Glucose DMEM and passed through a 70 um strainer to make a single-cell suspension. The resulting pellet was resuspended in 1X DPBS + 0.1% BSA + 0.5% EDTA. Suspension was incubated with antiobodies against SSEA1 (ovarian and testicular PGCs) and CD31 (ovarian PGCs only) at 4C for 30 minutes (ovarian PGCs) or 20 minutes (testicular PGCs). Cells were pelleted by centrifugation and resuspended then loaded onto an MS column on a MiniMACS magnet. After incuibation 1 mL of 0.1% BSA, 0.5% EDTA and 1x DPBS was loaded onto the column and cells released using the supplied plunger. After isolation cells were washed in 1x DPBS and resuspended in 1% paraformaldehyde at room temperature for 10 minutes. Fixation was stopped with 0.14M Glycine in DPBS and incubated rotating at room temperature for 10 minutes. Cells were pelleted by centrifugation, supernatant removed and flash frozen in liquid nitrogen. After thaw, cells were resuspended in 10 mM Tris-HCl, 0.25% Triton-X, 10 mM EDTA, 0.5 mM EGTA and Roche cOmplete and incubated at room temperature for 15 minutes. Cells were pelleted by centrifugation at 4000 RPM for 5 minutes. Pellets were resuspended in 10 mM Tris-HCl, 200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, and Roche cOmplete for 10 minutes rotating at 4C. Cells were pelleted by centrifugation at 4000 RPM for 5 minutes. Resulting pellet was resuspended in 10 mM Tris-HCl, 10 mM EDTA, 0.5 mM EGTA, Roche cOmplete. Resuspended cells were sonictaed using a Covaris S2 with the following program: Intensity = 5, Cycles/burst = 200, duty cycle = 5% with a 30" on/30" off cycle for an effective sonication time of 4 minutes. Sonicated lysate was centrifuged at 14000 rpm for 10 minutes at 4C. 10% of clarified lysate was held for input. Samples were pre-cleared with Protein A Dynabeads. Beads were removed at antibody added to sample to incubate, rotating, overnight at 4C. Protein A dynabeads were then added to the sample and left to incubate for 2 hours at 4C. Dynabeads were seperated via magenetic separation. Washed Dynabeads were then resuspended in 50 mM Tris-HCl, 1 mM EDTA, 1% SDS for elution on a thermoshaker at 65C, shaking at 1400 rpm for 10 minutes. Dynabeads were seperated and the elution was performed again on sperated beads. Input and eluted chromatin from above were then incubated overnight at 65C. Samples were treated with RNAse A for 30 minutes at 37C and then proteinase K at 56C for 2 hours. DNA was eluted using a MinElute column. Library was prepared using Tecan Genomics UltaLow System V2 according to manufacturer's instructions.