TRPS1 (self-made affinity-purified antibody raised against aa 920-1281)
Sequenced DNA Library
Cells were cross-linked with 1% formaldehyde for 10 min at RT. Nuclei were extracted using hypotonic buffer (20 mM Tris, pH 7.4, 2 mM MgCl2, 5% glycerol) and lysed with ChIP lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 1% NP40, 0.5% DOC, 0.1% SDS, 1 mM EDTA). Chromatin was sonicated to obtain fragments of 200 bp. Equilibration of beads (Dynabeads, Thermo Scientific) with antibodies (2 μg for ChIP, 10 μg for ChIP-Seq) was performed overnight, following immunoprecipitation of chromatin for 6h. After extensive washing, bound chromatin was eluted using ChIP elution buffer (50 M Tris, pH 8.0, 1 mM EDTA, 1% SDS, 50 mM NaHCO3) and de-crosslinked and RNase A/Proteinase K (Roth) digested over night. DNA was purified by phenol/chloroform extraction and ethanol precipitation. ChIP-Seq libraries were generated according to the manufacturer's protocol (NEBNext Ultra II DNA Library Prep Kit for Illumina, NEB) using Dual Index Primers (NEBNext Multiplex Oligos for Illumina, NEB). The number of PCR cycles varied between 9-12 PCR cycles. The optimal PCR cycle number was determined beforehand using a qPCR machine with 10% of the material used for the final amplification.