GSM8633803: Mouse_Cortex_bulk_nucleosome_depletion; Mus musculus; OTHER
Damagenome libraries were diluted to 35 μl with 1× NIB-Tris buffer. Then, 5 μl of 4 M NaCl, 5 μl of 10% SDS (Invitrogen, 15553-035) and 5 μl of 10 mg ml−1 Protease K (NEB, P8107S) were added and nuclei were lysed at 850 r.p.m. at 55 °C for 2 hours in a ThermoMixer. The lysed solution was cooled to room temperature and then purified with 1× SPRI beads (Beckman Coulter, B23319) and eluted in 12.5 μl of H2O. The formaldehyde fixed cells are collected and re-suspended in ice cold NIB buffer for nucleosome depletion; the DNA oxidative damage sites were treated with enzymatic labeling with biotin and tagmentated with identical barcoded assembled pA-Tn5, nuclei were lysed and gDNA and RNA were purified with 1X SPRI beads; next, DNA libraries were amplified by Kapa HiFi uracil plus DNA polymerase and used 0.8X Ampure XP beads to purify the product. The final libraries were sequenced using Nova-seq platform (illumina) with the following read lengths: PE 100 + 8 + 8 + 50 (Read 1 + Index 1 + Index 2 + Read 2).