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Library Info Page


Library ID

Library Name:Mouse Organ of Corti cDNA pBluescript
Organism:Mus musculus
Strain:BALB/c
UniGene Library ID:10920
Tissue Description:Organ of Corti, mouse, cell types: inner and outer hair cells, Deiters cells, Hensen cells, inner and outer phalangeal cells. Maybe some neuronal cells, fibroblasts and blood cells. 364 pooled samples.
Library Keywords:normal, bulk, juvenile, EST, MGC, mixed pool by gender, plasmid vector, organ of Corti

Clones and Sequences

#Clones Generated to Date:1536
#Sequences Generated to Date:7547

Library Preparation Details

Description:The organ of Corti (OC) was fine dissected from a total of 386 OC as follows: 102 samples from post-natal (P) day 5; 72 from P6; 60 from P7; 46 from P8; 18 from P9; 20 from P10; 14 from P12 and 24 from P13. After killing animals by cervical dislocation followed by decapitation, the bulla was removed and opened in Leibowitz medium. The bony capsule of the cochlea was chipped away, stria vascularis and spiral ligament were removed and the sensory epithelium was carefully dissected out of the modiolus. Total RNA was extracted using the micro Fasttrack kit (catalog # K1593-02; Invitrogen, Carlsbad, CA), according to manufacturer's instructions. Reverse transcription and library construction were carried out with the Uni-Zap XR vector kit (catalog # 237211, Stratagene) and Uni-Zap XR Gigapack III Gold Cloning kit (catalog # 237612), both from Stratagene (La Jolla, CA, USA), according to manufacturer's instructions. Briefly: 1.5 ug mRNA was reverse transcribed using a hybrid oligo(dT) linker-primer that contains an Xho I site. First strand synthesis was primed with the linker- primer and transcribed using Moloney murine leukemia virus reverse transcriptase (MMLV-RT) and 5-methyl dCTP. The second strand was synthesized with DNA polymerase and RNase H. Complementary DNA was blunt ended with Pfu DNA polymerase, ligated with EcoR I adapters in the presence of ligase and digested with Xho I. The cDNA was sequentially size fractionated over Pharmacia Size Sep400 (Pharmacia, Uppsala, Sweden) and Clontech Chroma Spin-1000 (Clontech, Palo Alto, CA) columns to enrich for cDNAs greater than 400bp and 1000 bp, respectively. The cDNA was then directionally ligated to the Uni-ZAP XR vector, which had been predigested with EcoR I and Xho I. The phagemid was packaged with Gigapak III Gold and, upon titration on XL1 Blue MRF' cells, the yield of the phage library was estimated to be 11,100,000 recombinants. Stratagene's ExAssist Interference resistance helper phage (catalogue # 211203) was adopted to rescue plasmid DNA from the phages. Upon plating of the rescued library, individual cDNA clones were selected and grown in 96-well, 2 ml growth plate. Plasmid DNA was purified from 200 ul of saturated culture with the Concert96(TM) plasmid purification kit (Invitrogen, Carlsbad, CA) as instructed by the manufacturer. ESTs from the 5' end of the cDNA clones were generated with the universal M13 reverse primer (CAGGAAACAGCTATGACC) and 25% strength BigDye terminator sequencing chemistry (Applied Biosystems, Foster City, CA). Sequencing reactions were performed on MJ Tetrad thermal cyclers (MJ Research, Waltham, MA), and analyzed on 3700 automated capillary sequencers using POP5 polymer (Applied Biosystems, Foster City, CA). The frequency distribution of the library is as follows: 72% of genes have 1 copy; 14.3% 2; 12% 3-10; 1.4% 11-50 and 0.1% 51-150. As to gene function, 45% of genes are present in GenBank and have know function; 23% have hits in GenBank, but do not have assigned function; 12% are uncharacterized ESTs and 20% are unidentified.
R. Site1:
R. Site2:
Lab Host:
Vector:pBluescript
Vector Type:Plasmid
Tissue Supplier:Bechara Kachar and Celine Pompeia, NIDCD.
Library Producer:Bechara Kachar, Celine Pompeia, NIDCD