MeDIP-Seq is a great way to detect DNA methylation anywhere in the genome with generation of less data as compared to bisulfite sequencing. A specific antibody for 5-methylcytosine is used to enrich methylated genomic DNA fragments, which are then used for sequencing. Unlike RRBS which only scans about 10% of CpGs, MeDIP-Seq, can cover regions all across the genome.
- Sequencing of entire genome is not necessary, therefore large data set generation is not required
- MeDIP-Seq coverage is not as biased toward high CpG density
- Submit 1-5 µg of RNA-free genomic DNA (NGS grade) of molecular weight >40kb, Nanodrop A260/280 ratio >1.8; A260/230=2.0-2.2; and at a concentration of ~50 ng/µl
- Or 5-10 g of fresh tissue fast frozen in liquid nitrogen and shipped in dry ice to eurofins facility.
- RNA free NGS grade gDNA isolation will be carried out using commercially available kit with further purification making it suitable for NGS library preparation.
- Further quality checking will be carried out using agarose electrophoresis and DIN value estimation using Agilent Tape Station. Quantification will be carried out using Qubit 3.0 fluorometer.
- Appropriate amount of gDNA will be mechanically sheared using covaris followed by illumina adaptor ligation and immunoprecipitation using 5-mC mAb. MeDIP Seq library will be prepared using TruSeq DNA methylation or TruSeq Nano DNA kit from illumina.
- Library validation will be carried out using High Sensitivity D1000 Screen tape using Agilent Tape station kit.
- Prepared library will be sequenced using 2 x 75 bp using NextSeq 500 or 2 x 125 bp using HiSeq 2500 using latest sequencing chemistries.
- Delivery of data up to ~ 4-5 Gb of data.
- Sequence data (base call files or bcl files) generated from the sequencer are de-multiplexed if required and converted to FASTQ files.
- Raw data will be available for download as a compressed archive of FASTQ files for each sample.
- Raw data can be post processed upon request
- 5-7 weeks after arrival of your samples and all necessary information. It depends upon data size, scope, technology selected, number of samples and complexity of the project. The TAT is offered expecting no biological or technical difficulties for processing of all project samples.