SRA

Third instar larvae were sexed and primary hemocytes collected from wild-type and Nurf301 mutant female third instar larvae. Larvae were ripped in batches of 50 third instar larvae into HyQ-CCM3 insect medium containing protease inhibitors. Cells were fixed with 1% formaldehyde in 1XPBS for 15 minutes at 25°C and pelleted at 400g for five minutes. Cells were washed three times with ice cold 1XPBS containing protease inhibitors and stored as pellets at -80°C until cells from 1000 animals had been collected. Hemocytes were thawed, pooled and digested with MNase to liberate mononucleosomes. Formaldehyde cross-links were removed by aProteinase K incubation at 65°C overnight. DNA was purified by phenol/chloroform extraction and ethanol precipitation, treated with RNase at 37°C for 1 hour and then run on 2.2% recovery FlashGels. Mononucleosomal DNA was pipetted from recovery wells in FlashGel Recovery Buffer and DNA subjected to further round of phenol/chloroform extraction and ethanol precipitation. Fragment libraries were generated from two biological replicates for each genotype, sequence on a SOLiD 4 analyzer, and reads pooled for nucleosome mapping.