IPSC_Smad3_rep1
Protocols: iPSCs were grown in 100mm plates and differentiated for 0 or 7 days by culturing in APEL2 medium supplemented with 8uM CHIR99021 for 3 days and APEL2 medium supplemented with 200ng/ml FGF9 for 4 days resulting in differentiation into nephron progenitor cells. Chromatin was prepared for shearing using a truChIP Shearing Kit according to manufacturer's protocol. 5 x 107 cells were fixed in 1X Fixing Buffer A containing 11.1% methanol-free formaldehyde for 5 minutes on an oscillating table. Crosslinking was quenched using Quenching Buffer E for 5 minutes. Fixed cells were scraped into ice cold DPBS and centrifuged at 500 xg for 5 minutes at 4°C. Cells were lysed using 1X Lysis Buffer B containing 1X protease inhibitors for 10 minutes at 4°C. Intact nuclei were collected by centrifugation, washed twice in 1X Wash Buffer C, and resuspended in 1X Shearing Buffer D3 containing 1X protease inhibitor. Chromatin was sheared in a pre-cooled AFA microtube using a Covaris E220 Evolution AFA focused-ultrasonicator for 20 minutes at duty factor of 2%, 200 cycles per burst, and peak incident power of 140.Protein G magnetic dynabeads were pre-blocked in 1 x PBS with 0.5% BSA before incubation with 3 µg Smad3 (Abcam; ab28379) or 2.5 µg EZH2 (Cell Signalling; 5246) antibodies on a rotator overnight at 4°C. Sheared chromatin was diluted four times in dilution buffer (140mM NaCl, 50 mM HEPES pH 8.0, 1mM EDTA, 0.75% Triton X-100, 0.1% Na-deoxycholate, 1X protease inhibitor cocktail) and mixed with 30 µl of dynabead-antibody mix. Samples were incubated overnight by rotation at 4°C. Beads were subsequently washed once with low salt buffer (20mM Tris-HCl pH 8.0, 150 mM NaCl, 2mM EDTA pH 8.0, 0.1% SDS), high salt buffer (20mM Tris-HCl pH 8.0, 500 mM NaCl, 2mM EDTA pH 8.0, 0.1% SDS), LiCl buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250nM LiCl, 1% IGEPAL), and twice with TE buffer containing 50 mM NaCl (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 50mM NaCl). Samples were eluted in 100 µl elution buffer (50mM Tris-HCl pH 8.0, 10 mM EDTA pH 8.0, 1% SDS) at 65°C. Samples were centrifuged at 16,000 xg for 1 minute at room temperature. Whole cell extract (WCE) to be used for input was diluted three times with elution buffer. Crosslinking was reversed in all samples by incubating at 65°C overnight. 100 µl TE buffer was added to each sample to dilute SDS in elution buffer. Reverse-crosslinked DNA was treated with RNase A and Proteinase K for 2 hours at 37°C and 55°C, respectively. DNA was purified using the QIAquick PCR purification kit as per manufacturer's protocols. DNA was eluted in 70 µl Buffer EB and stored at -20°C until use. DNA concentrations were quantified us a Qubit dsDNA high-sensitivity kit. ChIP libraries were prepared using the Illumina TruSeq ChIP sample preparation kit as per manufacturer's protocols.