title: SIPPE Gossypium hirsutum 5K cDNA fiber and ovule array
status: Public on Apr 12 2006
contact.city: Shanghai
contact.first: Xiao-Ya
contact.last: Chen
contact.email: xychen@sibs.sc.cn
contact.address: No. 300, Fenglin Road
contact.department: Institute of Plant Physiology and Ecology
contact.country: China
contact.web_link: http://xy.sippe.ac.cn/
contact.institute:  Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences
contact.phone: 86-21-54924033
organism: Gossypium hirsutum
description: To construct a cotton cDNA library, total RNA was isolated from the -3- to 3-DPA ovules and 6- to 24-DPA fibers by a cold-phenol method as previously described (Li, et al. 2002). Poly (A)+RNA was purified by an Oligotex mRNA Midi kit (QIAGEN, Valencia, CA) according to the manufacture’s instruction, and cDNA libraries were constructed following the ZAP-cDNA Synthesis kit (STRATAGENE，La Jolla, CA). The cDNA inserts were sequenced with T7 primer by ABI3700 automatic Sequencer. The ESTs have been deposited into NCBI database with accession number from DT_046365 to DT_054205.
manufacture_protocol: Based on cluster and annotation, 5122 putative unique EST clones were selected and their inserts were amplified by PCR, using T3 and T7 primers. House-keeping histones (H2A, H2B, H3, and H4) were included as internal controls. After purification and quantification, the PCR products plotted onto the glass slides (Full Moon BioSystems, Sunnyvale, CA) by GeneMachines OmniGrid Microarrayer (BST Group，Singapore).  Probes were prepared by the MICROMAX TSA Labeling and Detection Kit (PerkinElmer, Wellesley, MA, USA) using 10 μg total RNA. The probes were annealed to target cDNAs for 13 hr at 60 C, and arrays were washed thrice for 20 min each: 60 C in 2×SSC/0.2SDS, room temperature in 2×SSC, and room temperature with 0.2×SSC.