GSM6139207: IgG control; Mus musculus; ChIP-Seq
GSM6139207
For Eya1 ChIP-seq, 80 kidneys from E13.5 Eya1HA-FLAG knockin mice were used for each ChIP assay. The kidneys were cross-linked with 1% formaldehyde at room temperature for 60 mins, and then homogenized and lysed in cold lysis buffer (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1´ protease inhibitors). Samples were pelleted at 2000 g at 4°C and resuspended in cold wash buffer (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1´ protease inhibitors) for 10 minutes in 15 ml conical tubes, followed by spinning at 2000 g at 4°C in a benchtop centrifuge. Samples were resuspended in 1 ml cold sonication buffer (10 mM Tris-Cl, pH 8.0, 2 mM EDTA, 0.1% SDS) and sonicated to 200-500 bp fragments using a Covaris S220 Focused-ultrasonicator. Sonicated chromatin was cleared by pelleting insoluble material at 13,000 RPM at 4°C, followed by preclear with protein A/G beads and incubation with 20 ml blocked anti-FLAG M2 beads at 4°C for another 5 hrs. Immunoprecipitated complexes were subjected to series of wash steps with low salt buffer (20 mM Tris-Cl 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), high salt buffer (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% sodium deoxycholate) and TE plus NaCl, followed by elution with Flag peptide and reverse crosslinking overnight at 65°C. The quality controls of ChIPed DNA was performed with Qubit 2.0 Fluoremeter using dsDNA HS assay Kit (Q32854, ThermoFisher Scientific) and Agilent 2200 TapeStation System using High Sensitivity D1000 Reagents (5067-5585, Agilent). The libraries for sequencing were prepared with the ThruPLEX DNA-seq Kit (R400429, Rubicon Genomics) and sequenced on Illumina NextSeq500.