Funding: A grant from the Monsanto Company to the University of Missouri. Genetic Source: Endometrium and oviduct tissues from various stages of the estrous cycle were collected from crossbred pigs (Sus scrofa domestica), frozen in liquid nitrogen immediately after collection, and stored at -80 degrees Celsius until RNA extraction. The specific tissues collected were Day 0 and Day 3 whole oviducts and Days 3, 6, 10 and 12-14 endometrium. More information regarding the methods can be found at: http://genome.rnet.missouri.edu/Swine/Methods.html. Library Construction (Standard Protocol): All procedures discussed in this section 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 poly(A)+ RNA was obtained by two rounds of purification with the Oligotex mRNA isolation kit (Qiagen) according to the manufacturer's instructions. The oviduct libraries and the Day 3, 6 and 10 endometrium libraries were constructed essentially as described by the manufacturer's instructions provided with the SuperScript Plasmid System (Invitrogen, cat. no. 18248-013). Briefly, 1mg of poly(A)+ RNA will be annealed at 37 degrees Celsius with 10mg of NotI-tag-dT18 oligonucleotide (GCTGCTCGCGGCCGC-tag-T18) and reverse transcribed at 37 degrees Celsius 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 was ligated to SalI adapters (Invitrogen-Life Technologies) and digested with NotI. The cDNAs will be 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 pSPORT1 vector (Invitrogen). After ligation of the inserts, the plasmids will be electroporated into DH10B bacteria. The day 12-14 endometrium library was synthesized by Dr. Bento Soares' laboratory (University of Iowa) and was cloned into the T3T7pac vector as described elsewhere (Bonaldo et al., 1996). 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. Bioinformatics work was performed by GK Springer's bioinformatics group (WG Spollen, JE Ries, A Guillen, AA Khambati, RV Patel, CM Topinka, SB Bhuiyan) 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: [email protected]. Citations: 1. Bonaldo MF, Lennon G, Soares MB. Normalization and Subtraction: Two approaches to facilitate gene discovery. Genome Res, 1996; 6:791-806. 2. 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. 3. 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.