characteristics.text: LIBEST_004045
characteristics.characteristic: External Id
characteristics.text: SAMN00158598
characteristics.characteristic: External Id
characteristics.text: 2014-02-03T23:44:10.107Z
characteristics.characteristic: INSDC first public
characteristics.text: 2015-05-20T14:25:19.767Z
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: Anchor enzyme: NlaIII~ Tissue supplier: LNCaP Cells from ATCC~Library Producer: Srivastava S/Xu L, CPDR, USUHS~LNCaP cells (Item Number CRL-1740, American Type Culture Collection, Rockville, MD) were used for generating SAGE libraries. LNCaP cells were maintained in RPMI 1640 (Life Technologies, Inc., Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS, Life Technologies, Inc., Gaithersburg, MD) and experiments were performed on cells between passages 20 and 30. For the studies of androgen regulation charcoal/dextran stripped androgen-free FBS (cFBS, Gemini Bio-Products, Inc., Calabasas, CA) was used. LNCaP cells were cultured first in RPMI 1640 with 10% cFBS for 5 days and then stimulated with an non-metabolizable androgen analog R1881 (DUPONT, Boston, MA) 10-8 M for 24 hours (for generating LNCaP-T library). LNCaP cells identically treated but without R1881 treatment served as control (for generating LNCaP-C library). Cells were harvested at indicated time and polyA+ RNA was double-selected with Fast Track kit (Invitrogene). SAGE libraries were generated according to the procedure provided by Dr. Kinzler (Johns Hopkins University School of Medicine, Baltimore, MD). Briefly, biotinylated oligo dT primed cDNA was prepared from five micrograms of polyA+ RNA from treated and control LNCaP cells and biotinylated cDNA was captured on strepravidin coated magnetic beads cDNA bound to the magnetic beads were digested by NlaIII followed by ligation to synthetic linkers containing a site for anchoring enzyme, NlaIII and a site for tagging enzyme BsmF1. The restriction digestion of ligated products with BsmF1 resulted in the capture of 10-11 bp sequences termed as tags representing signature sequence of unique cDNAs. Strategy combining ligation, PCR, enzymatic digestion and gel purification yielded two tags linked together termed as ditags. Ditags were concatamerized, purified and cloned in plasmid pZero cloning vector (Invitrogen, CA). The clones containing concatamers were screened by PCR and sequenced.
characteristics.characteristic: description
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: NlaIII
characteristics.characteristic: re_1
characteristics.text: SAGE_CPDR_LNCaP-T
characteristics.characteristic: title