ChIP Sequencing

Chromatin Immunoprecipitation (ChIP) coupled with next generation sequencing as a detection method (ChIP-seq) has become one of the primary methods to discover genome wide protein-DNA interaction. This technique is now used in a variety of life science disciplines including cellular differentiation, tumor suppressor gene silencing, and the effect of histone modifications on gene expression.

 

In ChIP-Seq, enriched DNA regions (protein binding sites) are detected as peaks above background reads, and bioinformatics analyses of these regions can reveal binding motifs. Applications include studies on gene regulation, transcription complex assembly, histone modification, developmental mechanisms, and disease processes.

The major steps in ChIP Sequencing are given below:

  • Chromatin preparation: cell fixation (cross-linking) of chromatin-bound proteins such as histones or transcription factors to DNA followed by cell lysis.
  • Chromatin shearing: fragmentation of chromatin by sonication down to desired fragment size (100-500 bp).
  • Chromatin IP: protein-DNA complexe capture using specific ChIP-seq grade antibodies against the histone or transcription factor of interest.
  • DNA purification: chromatin reverse cross-linking and elution followed by purification.
  • NGS Library Preparation: adaptor ligation and amplifcation of IP’d material.
  • Sequencing: Sequencing on Illumina platform, generally single-end 50bp read length is suitable for ChIP Sequencing.
  • Bioinformatic analysis: read filtering and trimming, read specific alignment, enrichment specific peak calling, QC metrics, multi-sample cross-comparison etc.

Whole Genome Bisulfite Sequencing

DNA contains combinations of four nucleotides which include cytosine, guanine, thymine and adenine. DNA methylation refers to the addition of a methyl (CH3) group to the DNA strand itself, often to the fifth carbon atom of a cytosine ring. This conversion of cytosine bases to 5-methylcytosine is catalysed by DNA methyltransferases (DNMTs).  These modified cytosine residues usually lie next to a guanine base (CpG methylation) and the result is two methylated cytosines positioned diagonally to each other on opposite strands of DNA.

 

DNA methylation at the C5 position of cytosine plays a crucial role in gene expression and chromatin remodelling, and perturbations in methylation patterns are implicated in the development of cancer, neurodegenerative diseases, and neurological disorders. Therefore, the mapping of methylated bases (the methylome) is critical to understanding gene expression and other processes subject to epigenetic regulation.

 

Nucleome provides whole genome sequencing of bisulfite-converted DNA, as an effective method to identify individually methylated cytosines on a genome-wide scale. Methylome analysis is an increasingly valuable research tool with a range of applications, including studies on gene regulation, stem cell differentiation, embryogenesis, aging, cancer and other diseases, and phenotypic diversity and evolution in plants and animals. We recommend Paired end 150 bp sequencing of 250~300-bp insert bisulfite treated DNA library on HiSeq system. We provide a comprehensive data analysis report which includes raw data QC, alignment, Motif analysis, DMR etc. For more information visit Bioinformatics analysis page. We also offer ‘Reduced Representation Bisulfite Sequencing’ to determine the methylation in economical way.