Bsh-Dam of L5 Drosophila
lamina were collected (age equivalent at 25C: 76h APF) in cold PBS within one hour and stored at -20C immediately until sufficient lamina were collected for each group, about 70 lamina from 35 pupae. The TaDa experimental pipeline was followed according to (Marshall et al., 2016) with a few modifications. Briefly, DNA was extracted using a QIAamp DNA Micro Kit (Qiagen), digested with DpnI (NEB) overnight, and cleaned up using Qiagen PCR purification columns. DamID adaptors were ligated using T4 DNA ligase (NEB) followed by DpnII (NEB) digestion for 2 h and PCR amplification using MyTaq HS DNA polymerase (Bioline). The samples were sequenced on the NovaSeq at 118 base pairs and 27-33 million single end reads per sample.