title: IOLR_Cherax quadricarinatus_5.755K_v1.0
status: Public on May 27 2010
contact.city: Haifa
contact.first: Moshe
contact.last: Tom
contact.email: tom@ocean.org.il
contact.address: P.O.B. 8030
contact.department: Marine Biology and Biotechnology
contact.country: Israel
contact.web_link: http://www.ocean.org.il/
contact.institute: Israel Oceanographic and Limnological Research
contact.phone: 972-4-8565-257
organism: Cherax quadricarinatus
description: The array was printed on 2.5 x 7.5 cm glass slides (GAPS II, Corning) on an area less than 6 x 2 cm. The grid system includes 4 x 12 sub-arrays, each containing 16 rows and 16 columns, a total of 12288 spots. The printing pattern included two complete and separate sets of the transcriptome, in the upper and lower part of the slide, respectively. Empty spots which include only the printing buffer were taken out of the data table but their position on the slide can be identified (designated "XX") in the GPR files accompanied each experiment-related dataset.
manufacture_protocol: X Organ – Sinus Gland complex was bi-laterally extirpated from several C. quadricarinatus males to induce molting initiation. The molt cycle stage was monitored by gastrolith size, maxillar setae appearance and in some individuals the presence of new cuticle under the old one. Gastrolith disks were dissected from sacrificed males 10 days post-extirpation. Several hepatopancreatic lobes, hypodermis layered beneath the carapace or new hypodermis already formed, muscle tissue and the entire Y organ pair were dissected from sacrificed males 13 or 14 days post-extirpation. RNA was extracted from the dissected tissues. Dissected muscle served as a reference organ, whereas the molt-related gastrolith disks, hepatopancreas, hypodermis and Y-organ were the target organs. The cDNA assemblage was constructed in two stages: (1) SMART (Switching Mechanism At 5' end of the RNA Transcript), aimed at amplification of reverse transcribed cDNAs using a universal primer, linked to both ends of the template cDNA during the reverse transcription stage (Clontech, manufacturer instructions). (2) SSH (Subtractive-Suppression Hybridization method; Diatchenko et al. 1999; PCR-select, Clontech), aimed at T-A cloning of a roughly equally represented cDNAs from the target organs into an expression plasmid (pGEM-Teasy, Promega) biased towards genes differentially expressed in the target tissues in comparison to the reference one. The cloned cDNAs were transfected into E. coli, and 5755 bacterial colonies were isolated, amplified and frozen at -70C. A subset of the clones were successfully sequenced and assembled by the CAP3 software (Huang and Madan, 1999), to identify unique sequences. All the 5755 clones were amplified by standard PCR from 1 µl bacterial culture templates (5’ at 95C; 32 cycles of 0.5’ at 95C, 0.5’ at 68C and 1.5’ at 72C; a final incubation of 4’ at 72C). PCR products were precipitated from the PCR solution by one volume of isopropanol at room temperature. The dry DNA was dissolved in printing buffer at final concentration of 50% DMSO and 0.3 x SSC and was printed on a glass slide (GAPS II slides, Corning; MGII robot-printer, BioRobotics) in duplicates. The amplification efficiency was tested by running the PCR products on a 1% agarose gel concurrent with a DNA mass ladder. The platform is a broadening of a previous platform designated by GEO accession number GPL4786 (Yudkovski et al., 2007), with updated annotations.
  
  References:
  
  Diatchenko, L., Lukyanov, S., Lau, Y.F.C. Siebert, P.D. (1999) uppression subtractive hybridization: A versatile method for identifying differentially expressed genes. In: cDNA Preparation and characterization, Meth. Enzymol. 303: 349-380.
  
  Huang, X. and Madan, A. (1999) CAP3: A DNA sequence assembly program. Gen Res 9: 868-877.
  
  Yudkovski, Y., Shechter, A., Chalifa-Caspi, V., Auslander, M., Ophir, R., Dauphin-Villemant, C., Waterman, M., Sagi, A. & Tom, M. (2007). Hepatopancreatic multi-transcript expression patterns in the crayfish Cherax quadricarinatus during the moult cycle. Insect Mol. Biol. 16, 661-674.