GSM5135480: DamID_freedam_set7_N7476; Neurospora crassa; OTHER
Preparation of N6-methyladenine-containing DNA for sequencing was performed using a previously reported procedure (Bernstein et al. 2012) with the following modifications: 5 μg of genomic DNA from N. crassa strains expressing a Dam fusion was digested with 1 μL of DpnI (NEB, 20 units/μL); ligation to primer 5050 was carried out overnight at 16 °C; amplification reactions of ligated DNA with primer 5051 were performed in triplicate, contained 5 μL dNTPs, and an additional PCR cycle was added to the 2nd and 3rd phase of the PCR protocol (4 and 18 cycles respectively); 3 μg of pooled, amplified DNA was sheared using a Bioruptor (Diagenode) twice on high for 10 minutes (30 seconds on/off) at 4 °C. Biotinylated DNA was purified using 250 μL slurry of streptavidin-agarose beads (Sigma) and prepared for high-throughput sequencing using the NEBNext DNA Library Prep Master Mix Set for Illumina (NEB).