Chromatin Immunoprecipitation (ChIP) combined with next generation sequencing (ChIP-seq) is a powerful experimental technique used to determine all the genomic locations of a specific protein-DNA interaction.
Do I need a negative control sample?
Yes! Without a negative control sample to determine the background noise, the results from the peak finding tools will determine a substantially higher number of false positives. There are a number of negative controls that can be used including:
- IgG - nonspecific immunoglobulin G
- Input – i.e. DNA that went through the process without any specific selection for fragments related to binding of transcription factors
- MockIP - usually obtained by performing all the steps in a ChIP protocol with the exception of antibody addition
More information on controls and setting up the experiment can be found in ChIP-Seq: technical considerations for obtaining high-quality data (Kidder et al, 2011), including a recommendation on using an input control over IgG:
"IgG may be less desirable in certain circumstances because of the following reasons: most IgG antibodies are not obtained from true preimmune serum from the same animal in which the specific antibody was raised; and IgG antibodies usually immunoprecipitate much less DNA than specific antibodies do, and thus limited genomic regions from the control may be over amplified during the library construction step"
Single reads or paired end reads?
Single read sequencing is usually sufficient for ChIP-seq analysis.
Data analysis pipeline:
- Quality assessment of the sequence reads: i.e. Fastqc
- Map reads to reference genome: BWA or Bowtie
- Determine ChIP binding regions: MACS
- Downstream analysis including annotation of peaks and intersection between different samples: Bioconductor ChIPpeakAnno package
- motif searching:
Bioconductor RGadem package
MEME-ChIP (part of the meme package)