Experimental Design

E    type of experiment ; expression profile of each tissues

E    experimental factors ; normal

E    the number of hybridizations performed experiment ; duplicate

E    the type of reference used for the hybridizations ; RNA from whole body of embryo 17.5 days

E    hybridization design ; cy3 : each tissue, cy5 : embryo 17.5 days

E    quality control steps taken ; duplicate, positive controls and negative controls

E    URL of any supplemental websites or database accession number ; http://READ.gsc.riken.go.jp/fantom2/

 

Sample used, extract preparation and labeling

E    the origin of the biological sample ; mouse ( C57BL/6J ), male, at 8week( adult ). the female specific reproductive organs were prepared from female mice at 8 week (adult).

E    manipulation of biological samples and protocols used ; Mice used in this study were bred under SPF condition. This experiment was approved by IACUC of RIKEN.

E    protocol for preparing the hybridization extract ; Total RNAs were extracted using the acid guanidine phenol chlorophorm (AGPC) method (Carninci, P. and Y. Hayashizaki. 1999. High-efficiency full-length cDNA cloning. Methods Enzymol 303: 19-44)

E    labeling protocol ; aminoallyl method

E    external controls ; positive controls (G3PDH, beta actin, elongation factor 2), negative controls (clones of Arabidopsis thaliana).

 

Hybridization procedures and parameters

E    the protocol and conditions used during hybridization, blocking and washing ; hybridization : 65Ž, over night, blocking : no blocking, washing : 2*SSC, 0.1*SDS -> 1*SSC -> 0.2*SSC

Measurement data and specifications

E    the quantifications based on the image ; DigitalGENOME (MolecularWare, Inc., Cambridge, MA, USA)

E    type of scanning hardware and software used ; ScanArray 5000 (GSI Lumonics Inc., Billerica, MA, USA)

E    type of image analysis software used ; DigitalGENOME (MolecularWare, Inc., Cambridge, MA, USA)

E    a description of the measurements produced by image-analysis software and a description of which measurements were used in the analysis ; Mean value of the pixels in the circled area (provided by the Manufacturer: MolecularWare, Inc., Cambridge, MA, USA)

E    the complete output of the image analysis before data selection and transformation ; available on request

E    data selection and transformation procedures ; Valid data were selected by the PRIM method (Kadota, K., R. Miki, H. Bono, K. Shimizu, Y. Okazaki, and Y. Hayashizaki. 2001. Preprocessing implementation for microarray (PRIM): an efficient method for processing cDNA microarray data. Physiol Genomics 4: 183-188.) The value of the ratio (Cy3/Cy5) was log base2 transformed and used.

E    final gene expression data tables used by the authors to make their conclusions after data selection and transformation ; available on request

Array design

E    general array design ; spotted glass array, gamma-amino-propyl-silane coated slides (CMT-GAPS coated slides, Corning, Inc., Corning, NY, USA)

E    For each feature on the array and the ID of its respective reporter (molecular present on each spot) should be given. ; available at http://READ.gsc.riken.go.jp/fantom2/

E    For each reporter, its type should be given ; RIKEN full-length enriched cDNA clones

E    along with information that characterizes the reporter molecule unambiguously, in the form of appropriate database references and sequence; All the sequences are available from the public database. The correlation of accession number is available at http://READ.gsc.riken.go.jp/fantom2/

E    For non-commercial arrays, the following details should be provided:

Ø       the source of the reporter molecules ; RIKEN full-length enriched clones

Ø       the method of reporter preparation ; CAP trapper method (Carninci, P. and Y. Hayashizaki. 1999. High-efficiency full-length cDNA cloning. Methods Enzymol 303: 19-44)

Ø       the spotting protocols ; the array substrate : PCR products, the spotting buffer : 3*SSC, post-printing processing : rehydration -> UV cross-linking

Ø       any additional treatment performed prior to hybridization ; blocking with 1-methyl-2-pyrrolidone and succinic anhydriden