MT10430(official name : MT10430 genotype : lin-35(n745) I. )
Sequenced DNA Library
Worms are grown on peptone-enriched plates seeded with E. coli (HB101) and maintained according to standard protocol. Briefly, starved and synchronized L1s are first obtained, and then plated on peptone-enriched plates with HB101, which serves as a food source. Worms are grown at 20ºC to the desired developmental stage before harvesting. Since growth rates are frequently strain-specific, we use developmental milestones to determine developmental stages. Worms at the designed developmental stage are immediately crosslinked with 2% formaldehyde, quenched with 100 mM Tris buffer, washed with M9 buffer, and then stored at -80 as packed pellets. The current ChIP Protocol is modified from Ercan et al., Nature Genetics 39: 403-408 (2007). Worm samples are lysed and solubilized by sonication in the presence of protease inhibitors and non-ionic detergents. The cellular debris is removed by centrifugation and the supernatant containing any formaldehyde crosslinked chromatin is saved for preparing input DNA and collecting immunocomplexes. 10% (or 50 ul) of the above supernatant from each sample is saved as input sample (control). Input sample (also known as whole cell extract) represents the total genomic DNA and is processed later (treated with RNAase A, proteinase K and reverse crosslink steps) along with the ChIPed samples to isolate genomic DNA (input DNA). The remaining supernatant is incubated with affinity-purified anti-GFP antibodies and Protein G-sepharose beads to collect the immunocomplexes containing a targeted transcription factor that is C-terminally tagged with GFP and its genomic binding fragments. Alternatively, the 8WG16 mouse monoclonal antibodies and Protein A-sepharose beads are used to collect the immunocomplexes containing RNA polymerase II. After extensive washing, any immunocomplexes are eluted and the protein-DNA crosslinks are reversed. The degree of sonication is assessed by running a small aliquot of DNA on a 2% agarose gel. Quantitative PCR is used to check if the ChIPed sample contains any of the targeted transcription factor's known genomic binding sites. DNA fragments recovered following chromatin IP are size selected using gel electrophoresis. The DNA is then prepared for deep sequencing using the protocols and reagents provided by Illumina. This involves rendering the ends blunt, followed by the addition of single deoxy adenylate residues on each end. The fragments are ligated to Illumina's propietary adapters and amplified. After a final gel purification step the DNA is loaded into a flow cell for sequencing.