title: Plasmodium falciparum 385K tiling array
status: Public on Sep 02 2009
contact.city: Nijmegen
contact.first: Richard
contact.last: Bartfai
contact.email: r.bartfai@ncmls.ru.nl
contact.address: Geert Grooteplein 26-28
contact.department: Molecular Biology
contact.country: Netherlands
contact.institute:  Nijmegen Center of Molecular Life Sciences
organism: Plasmodium falciparum
description: ChIP on chip:
  Chromatin immunoprecipitations were carried out using formaldehyde crosslinked chromatin isolated from synchronized trophozoites (3D7/PfHP1-HA). After formaldehyde crosslinking and saponin lysis, nuclei were separated using a 0.25M sucrose buffer cushion and sheared by sonication in a Bioruptor UCD-200 (Diagenode) for 15min at 30sec intervals (size range 100-500bp). 400-500ng DNA-containing chromatin was incubated with 1ug antibody (anti-HA, ab9110); in presence of 10ul A/G sepharose beads (Santa Cruz Biotechnology) overnight at 4°C. After extensive washes immunoprecipitated chromatin was eluted with 1% SDS and 0.1M NaHCO3 and de-crosslinked at 65°C for 4hrs. DNA was purified using PCR purification columns (Qiagen). For genome-wide analysis 24 individual ChIP reactions were combined using formaldehyde-crosslinked material. Immunoprecipitated DNA was amplified by a modified T7 linear amplification method (A. Salcedo-Amaya et al, PNAS, 2009). Briefly, DNA fragments were dephosphorylated using calf intestinal alkaline phosphatase (NEB) and subsequently G-tailed with terminal transferase (NEB). T7 promoter was incorporated using Klenow polymerase (NEB) and T7C9B primer. RNA was synthesized by T7 polymerase (Ambion T7 megascript kit) and subsequently reverse transcribed using N6 primers. Amplified dsDNA was labeled with Cy3- (ChIP) or Cy5- (input) coupled random heptamers and hybridized to a tiling array at Roche NimbleGen.
manufacture_protocol: DESIGN_ID	DESIGN_NAME	DESCRIPTION
  1992	2005-05-10_Plasmodium_3D7_WG_CGH	Plasmodium genome (3D7) tiled at 45 bp spacing. Probes selected at 4 bp primary interval with a 4 bp secondary interval. 10 probes selected at each primary interval and then uniqueness, frequency and basic rule scoring determined. Instead of normal Tm tiling, probes were allowed to grow as long as possible given the 148 synthesis cycle cutoff. Rank selection done for each primary interval 'cluster'. The 478402 probes selected by the rank selection were then filtered further: rules >= -300; frequency <= 25; tm: 60-80. This left 385585 probes for the design. Also added 2883 random tm70 probes with the following parameters: tm 70; 148 cycles; min length: 45; max length: 85; gc: 23%.