Anti-RNA Polymerase II antibody, CTD (clone 8WG16)
chip antibody vendor
EMD Millipore
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Total RNAs were extracted by RNeasy Plus Mini kit (Qiagen) according to the manufacturer’s protocol. RNA-seq libraries were generated using the NuGEN Encore Complete RNA-seq Library Systems (Part No. 0311, NuGEN). Chromatin immunoprecipitation were performed using approximately 1x10^7 cells of human iPSCs, fibroblasts, endothelial cells, and cardiac progenitor cells. Cells were first cross-linked with 1% formaldehyde for 10 min at RT, and formaldehyde was quenched by glycine with a final concentration of 0.125 M. Chromatin was broken into small pieces with an average size of 0.5-2 kb using the Bioruptor (Diagenode). The sonicated chromatin was then incubated with 3-5 μg of primary antibodies overnight at 4°C. A small portion (10%) of chromatin without antibody incubation was kept as input DNA for each ChIP reaction. Subsequently, 75 μl of Dynabeads Protein A or Protein G were added and incubator for 4 h at 4°C with overhead shaking. Magnetic beads were then washed away and chromatin was eluted. Crosslink was then reversed and precipitated DNA was purified and resuspended in nuclease-free water. Sequencing libraries of immunoprecipitated DNA and input DNA were constructed according to an Illumina DNA library preparation protocol.