DamID in intestinal tissue of young adults of C. elegans hermaphrodites to decipher the binding target sites for LET-418 and MEP-1
DamID in intestinal tissue of young adults of C. elegans hermaphrodites to decipher the binding target sites for LET-418 and MEP-1
C. elegans strains were grown on a lawn of GM48 (-dam) E.coli bacteria on NGM plates at 20°C for at least two generations. After synchronization, around 1500 L1 larvae were seeded on three 100 mm plates. The larvae were grown till the young adult stage and were collected after washing 10-12 times with M9 media in aliquots of 30-50 µl. After the removal of excess liquid, samples (approximately 4500 worms per sample) were snap frozen and stored at -80°C until further use. The list of strains used in the study is mentioned in the supplementary files DNA extraction from frozen worm pellets was performed by using the DNeasy Blood and Tissue Kit (#69504; Qiagen, Valencia, CA).  500 ng of DNA was used for every sample for digestion with dpnI followed by ligation of the adaptors and PCR amplification to generate DNA amplicons of each sample. For the intestinal DamID 18-22 PCR cycles were performed (sup Figure 1B, representative replicate 1). DNA amplicons purifications were performed after every step from the PCR amplification onwards using magnetic beads and DNA quantification was done using QUBIT. Library preparation included end repair, barcoding and pooling of the libraries to be loaded on sequencer.