characteristics.text: LIBEST_015389
characteristics.characteristic: External Id
characteristics.text: SAMN00149807
characteristics.characteristic: External Id
characteristics.text: 2014-02-03T23:44:04.303Z
characteristics.characteristic: INSDC first public
characteristics.text: 2015-05-20T14:15:14.187Z
characteristics.characteristic: INSDC last update
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: Funding: The production of ESTs submitted in this project was funded by USDA Grant MRI-2002-03476 entitled 'Bovine ESTs: Focus on Female Reproduction' to RS Prather, E Antoniou, HA Garverick, JA Green, MC Lucy, RM Roberts, MF Smith and RS Youngquist. Genetic Source: Heifers for the project were purchased from Circle A Ranch, Iberia, MO (http://www.circlearanch.com/home.html). These heifers, while not registered have known Angus pedigrees going back at least 4 generations. Samples collected: The samples consisted of the following: germinal vesicle-stage oocytes; in vitro derived embryos (2-cell, morula, blastocyst and nuclear transfer blastocyst); in vivo blastocysts and conceptuses (days 8, 14, 16 and 18); corpora lutea (days 3, 5, 8, 14, 16, 18 and 35); ovarian follicles (days 0, non-recruited, recruited, early selected and preovulatory); oviduct (days 0, 3 and 5); endometrium (days 5, 8, 14, 16, 18 and 35); and placenta/embryo from day 35 conceptuses. Expanded descriptions of how the tissues were collected can be found at the following URL: http://genome.rnet.missouri.edu/Bovine/Methods.html. Library construction (Standard Protocol): All procedures have been described in detail elsewhere (Soares et al., 1994; Bonaldo et al., 1996; Jiang et al., 2001). Total cellular RNA from each sample was isolated by using STAT-60 reagent (Tel-Test, Friendswood, TX) and the poly(A)+ RNA was obtained by two rounds of purification with the Oligotex mRNA isolation kit (Qiagen) according to the manufacturer's instructions. The libraries were constructed essentially as described by the manufacturer's instructions provided with the SuperScript Plasmid System (Invitrogen, cat. no. 18248-013). Briefly, 1mcg of poly(A)+ RNA was annealed at c37 degrees with 10mcg of NotI-tag-dT18 oligonucleotide (GCTGCTCGCGGCCGC-tag-T18) and reverse transcribed at c37 degrees with SuperScript II (Invitrogen) reverse transcriptase (Jiang et al., 2001). The 'tag' represents a tissue/stage-specific ten-base sequence identifier (http://genome.uiowa.edu/pubsoft/software.html) present in the oligonucleotide used to prime first-strand synthesis. Second strand synthesis was performed with T4 DNA polymerase in the presence of DNA ligase and RNase H. After second strand synthesis, the double-stranded cDNAs were ligated to SalI adapters (Invitrogen-Life Technologies) and digested with NotI. The cDNAs were size selected by passage through cDNA size fractionation columns (Invitrogen-Life technologies). The cDNAs derived from each developmental stage of a particular tissue were mixed on an equimolar basis and ligated directionally into the NotI and SalI sites of the pCMV-SPORT6 vector (Invitrogen). After ligation of the inserts, the plasmids were electroporated into DH10B bacteria. 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 the standard protocol. Therefore, PCR-based protocol was utilized for producing libraries from sources in which the amount of extracted mRNA was small (oocytes and embryos). 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 Chroma Spin-1000 columns (Clontech). Purified cDNA from each PCR reaction was quantitated and mixed on an equimolar basis for ligation 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 (~4 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. After production of the libraries, equal numbers of recombinants from each library were pooled to produce a single mixed library (mega-library) for more extensive sequencing. Bioinformatics work was performed by GK Springer's bioinformatics group in Computer Science 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: bovine@rnet.missouri.edu. Bonaldo MF, Lennon G, Soares MB,. Normalization and Subtraction: Two approaches to facilitate gene discovery. Genome Res, 1996; 6:791-806. Jiang H, Bivens NJ, Ries JE, Whitworth KM, Green JA, Forrester LJ, Springer GK, Didion BA, Mathialagan N, Prather RS, Lucy MC (2001) Constructing cDNA libraries with fewer clones that contain long poly(dA) tails. Biotechniques 31:38-42. Soares MB, MF Banaldo, P Jelene, L Su, L Lawton, A Efstrantiadis. 1994. Construction and characterization of a normalized cDNA library. Proc Natl Acad Sci, 91:9228-9232.
characteristics.characteristic: description
characteristics.text: Bos taurus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9913
characteristics.characteristic: organism
characteristics.text: bemiv_original
characteristics.characteristic: title