Early origins of replication

Early Origins of Replication (MacAlpine project, MacAlpine subgroup)

General Description

Early origins of replication were identified by treating cells with hydroxyurea (HU), a potent inhibitor of nucleotide synthesis, in the presence of the nucleotide analogue BrdU. Treatment of synchronized cells with HU stalls replication forks andactivates the intra S-phase checkpoint, thereby limiting BrdU incorporation to those sequences immediately adjacent to earlyactivating replication origins. BrdU enriched sequences surrounding early origins of replication are then enriched by immunoprecipitation with an anti-BrdU antibody. Early origins are then detected by hybridization to Agilent genomic tiling arrays.

Protocols

  1. Growth and isolation: cell_growth
  2. Sample preparation: brdu_labeling, Sequenase_labeling, MacAlpine_Array_Hybridization, MacAlpine_Array_Scanning
  3. Other Protocols: brdu_ip, MacAlpine_Data_Normalization_ma2c, MacAlpine_ma2C peak calling

Reagents

  1. Animals: Drosophila melanogaster
  2. Arrays: http://wiki.modencode.org/project/index.php?title=Array:Agilent_Dm_5.1_470_tiling:DM:1
Release Date: 2009-01-30