title: Origin of replication for Pyrococcus abyssi by ChIP-chip
status: Public on Jan 30 2007
contact.city: Gif-sur-Yvette
contact.first: LAWRENCE
contact.last: AGGERBECK
contact.email: mucchiel@cgm.cnrs-gif.fr, aggerbeck@cgm.cnrs-gif.fr, delacroix@cgm.cnrs-gif.fr
contact.address: Avenue de la Terrasse
contact.department: Centre de Génétique Moléculaire
contact.country: France
contact.institute: Centre National de la Recherche Scientifique
contact.phone: 33-(0)169823752
organism: Pyrococcus abyssi
description: Chromatin immunopreciptation coupled with hybridization on a whole genome microarray (ChIP-chip) of P. abyssi.
manufacture_protocol: Microarray preparation.
  The P. abyssi genome was divided into 353 regions, each of which is 5 kb in length. Each of the 353 DNA regions has a specific ID number. For example, the microarray region 25 corresponds to the genome coordinates 120 000 to 125 000. For the microarrays, each of the 353 regions was amplified with the BD Advantage 2 PCR kit (BD biosciences clontech). The purified PCR products were quantitated and distributed into 384-well source plates at final concentrations of 100 ng/µl in final volumes of 8 µl of water. PCR products were dried using centrifugation under vacuum and then resuspended in 8 µl of 1% CHAPS and 50% formamide (28).
  The DNAs were arrayed onto activated GAPSII slides (Corning) using a GeneTAC G3 robot (Genomic Solutions) operating under a controlled environment (20°C and 35% humidity) using thirty-two 150 µ diameter pins. Each fragment was spotted 6 times (3 spots in two different blocks originating from two different pins). P. abyssi plasmid pGT5 and negative controls (lambda DNA, pBluescript, pESP1, 1kb ladder) were also added after linearization by appropriate restriction enzymes. After spotting, the slides were dried overnight, protected from light, at room temperature. The DNA was then UV-crosslinked to the slide using 250 mJ/cm2 (Hoeffer UVC500; Amersham Biosciences). For quality control, one slide from every print batch was stained with SYBR Green II (Invitrogen) diluted 1:10 000. The slides were pre-processed by washing for 5 min each with 1) 2X SSC/0.1% SDS, 2) 0.2X SSC and 3) water and then dried using centrifugation under vacuum. Identical pre-processing was used for slides destined for hybridization.