Chromatin immunoprecipitation (ChIP) was carried out as previously described (16 (link)). For immunoprecipitation, antibodies against H3K27Ac (ab4729, Abcam), SMAD3 (ab28379, Abcam) or RNAPII (sc899, Santa Cruz) were used in this paper. Quantitative analysis of the precipitated and input DNA through real-time PCR was described above. The primer sets for ChIP-qPCR are listed in Supplementary Table S1 . Results are pooled from at least three individual samples. For ChIP-seq, the precipitated DNA was followed library generation (TruSeq ChIP Library Preparation Kit, Illumina) and sequencing (NextSeq500 system, Illumina) performed by DNA Link, Inc. The generated reads were aligned to the human reference genome (hg19) using MACS2 (30 (link)). BAM files for ChIP-seq data of RNAPII in multiple cell lines were downloaded from ENCODE (https://www.encodeproject.org/ ).
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Chromatin Immunoprecipitation and Analysis
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Antibodies
Cell lines
Chromatin immunoprecipitation
Genome human
Immunoprecipitation
Library
Primer
Real time pcr
Rnapii
Smad3
Corresponding Organization : The University of Texas Southwestern Medical Center
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Variable analysis
independent variables
- Antibodies used for immunoprecipitation: H3K27Ac (ab4729, Abcam), SMAD3 (ab28379, Abcam) or RNAPII (sc899, Santa Cruz)
- Precipitated DNA quantified through real-time PCR
- Precipitated DNA used for library generation and sequencing
- Chromatin immunoprecipitation (ChIP) protocol
- Human reference genome (hg19) used for read alignment
- MACS2 used for ChIP-seq data analysis
- ENCODE datasets of RNAPII ChIP-seq in multiple cell lines
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