GSM6808178: Micro-C Replicate1_Chd1; Saccharomyces cerevisiae; OTHER
The chromatin was reverse cross-linked with the addition of 1 mg/mL Proteinase K (Life Technologies) and 0.5% SDS at 65ºC for ≥3 hours. For DNA extraction, the reaction was supplemented with glycogen and subjected to ethanol precipitation. The ligation products were assessed using Fragment Analyzer. A successful ligation is indicated by the increase in fragment sizes of > 320 bp. Unligated mono-nucleosomal fragments were further removed by purification with 0.9 volume of AMPure XP beads. Approximately 150-200 ng of each library was sonicated to a mean fragment size of 200 bp using a Covaris S220 Ultrasonicator (peak incident power 175; duty factor 10%; cycles per burst 200; treatment time 250s). Bead cleanup with AMPure XP beads was then performed and the fragment size was assessed using Fragment Analyzer. Libraries were indexed using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs) using 170-200 ng input. The manufacturer's protocol was followed with the following deviations: To maximize library complexity and yield, the PCR was performed in duplicate per ligation reaction using Herculase II reagents (Agilent Technologies). The parallel library preparations and PCR reactions were subsequently pooled for each reaction. The quality of the indexed library and molar concentration were measured using Fragment Analyzer and Qubit. in vitro Micro-C