BioSample

Funding: A grant from the Monsanto Company to the University of Missouri. Genetic Source: Slaughterhouse-derived oocytes were collected, and after cumulous cell removal were used for germinal vesicle stage oocytes, or were matured in vitro (with cumulus cells attached), in vitro fertilized and cultured. In vivo produced 4-cell and blastocyst stage embryos were collected on days 3 and 6, respectively. Zonae Pellucidae were removed from the embryos prior to mRNA isolation. Expanded descriptions of how the tissues were collected can be found at the following URL: http://genome.rnet.missouri.edu/Swine/Methods.html. Library Construction (PCR Protocol): The amount of mRNA that was recovered from oocytes and embryos was quite limiting and was not sufficient for library production with a standard protocol. Therefore, PCR-based protocol was utilized for producing libraries. Poly-A RNA was isolated by using the MicroPoly(A) Pure kit from Ambion (cat. #1918). The mRNA was reverse transcribed with a NotI-tag-dT18 oligonucleotide and a SMART oligonucleotide (Clontech) modified to contain a SalI site to generate full-length cDNA with a sequence complementary to the SMART oligonucleotide. Sequences within the SMART and dT oligonucleotides were used as primers to amplify the cDNAs by PCR with pfu turbo polymerase (Stratagene). The resulting PCR products were purified, digested with NotI and SalI and size fractionated by using a Chroma Spin-400 followed by a Chroma Spin-1000 column (Clontech). Purified cDNA from each PCR reaction was ligated into the pCMV-SPORT6 vector. Preliminary Library Characterization: Randomly chosen clones from each library were analyzed by restriction digestion to determine average insert size (96 clones) and by sequencing (~3 96-well plates) to confirm library quality [e.g. the presence of short polyA+ tails, genomic DNA contamination (must be <1%), ribosomal RNA clones (must be <1%), etc.] and to provide a sequence database representing the predominant clones in each library. The clones were sequenced at the University of Missouri-Columbia DNA Core Facility. Bioinformatics work was performed by GK Springer's bioinformatics group (WG Spollen, JE Ries, A Guillen) in Computer Science and Health Management and Informatics Departments at the University of Missouri-Columbia. Clone Requests: Requests for clones should be made to the Director of the University of Missouri DNA Core facility at: porcine@rnet.missouri.edu.