GSM2213944: Dam repl 1; Drosophila melanogaster; OTHER
gDNA from 5*106 cells was isolated using ISOLATE II Genomic DNA Kit (Bioline). 500ng gDNA were digested with DpnI (10 U, New England Biolabs) in CutSmart buffer in a total volume of 20 µl at 37°C for 8 h. Reaction was terminated by heat-inactivation at 80°C for 20 min. Fragments were ligated to 12.5 pmol DamID adapters using T4 ligase (2.5 U, New England Biolabs) in T4 ligase buffer in a total volume of 25 µl incubated at 16°C for 16 h. The reaction was heat-inactivated for 10 min at 65°C. Products were then digested with DpnII to remove partially methylated fragments. DpnII buffer and DpnII (10 U, New England Biolabs) were added in a total volume of 80 µl and incubated at 37°C for 1h. 20µl of DpnII-digested products were amplified by PCR. PCR products were cleaned up using the ISOLATE II PCR and Gel Kit (Bioline) according to the manufacturer’s instructions and eluted in 26µl H2O. Fragment ends were blunted using the End-It™ DNA End-Repair Kit (Epicentre) in a 50µl reaction according to the manufacturer’s instructions. Products were cleaned up using the ISOLATE II PCR and Gel Kit (Bioline) and eluted in 26µl H2O. For adding a 3’ overhang, fragments were treated with Klenow fragment (3’-5’ exo-, 25 U, New England Biolabs) in buffer 2 with 200µM dATP (Thermo Fisher Scientific) in a 50 µl reaction incubated at 37°C for 30 min. Reaction was terminated by heat-inactivation at 75°C for 20 min. Products were purified using CleanPCR magnetic beads (CleanNA) according to the manufacturer’s instructions and eluted in 20 µl H2O. 260 ng fragments were ligated to 2.5 µM Y-adaptors using T4 Ligase (2.5 U, New England Biolabs) in T4 buffer in a total volume of 10 µl incubated at 16°C for 16 h. Reaction was terminated by heat-inactivation at 65°C for 10 min. Products were purified using CleanPCR magnetic beads (CleanNA) according to the manufacturer’s instructions and eluted in 20 µl H2O. Adapters and indices for next-generation sequencing and multiplexing were added by PCR.
GEO Accession
GSM2213944