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Protocols.
[3'RACE]
[Cloning]
[Bioinformatics]
[Website]
3'RACE
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3'RACE.
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| Total RNA was prepared from C.elegans samples at mixed developmental stages using the RNeasy kit (Qiagen, cat. # 74104). The total RNA is then used as a template for the first strand reaction with the SuperScript III Reverse Transcriptase from (Invitrogen, cat. # 18080-085) according to the manufacturers specifications. A transcript-specific forward primer, which anneals to a few nucleotides 5' to the STOP codon is used for each reaction. A unique anchored poly-dT reverse primer is used for all the reactions. Both primers contain sequences compatible with the Gateway system (Invitrogen). |
Cloning
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Cloning.
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Each RT-PCR product is cloned into the Gateway P2R-P3 vector (Invitrogen, cat. # 12537023) using the Gateway BP Clonase II enzyme mix according to the manufacturers protocol. Following the reaction, the recombined vectors are transformed into Multishot StripWell TOP10 chemically competent cells (Invitrogen, cat. # 44-0091) according the manufacturers directions. The transformed bacteria are grown overnight under antibiotic selection. The following day, 2 l of the bacteria is used as template for a PCR reaction with vector-specific Forward and Reverse primers. 5 l of each minipool is loaded on e-gel 96 2% agarose (Invitrogen, cat. # G7008-02) and several microliters are sent for sequencing to Agencourt Bioscience Corporation (www.agencourt.com).
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Bioinformatics Pipeline
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BioInformatics Pipeline.
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ABI Trace Files, and Sequence Traces are parsed using ad-hoc perl scripts developed in our laboratory at the Center of Comparative and Functional Genomics at New York University. We assign to each Trace file an internal score, which reflects the confidence that the trace contains 3'UTR data. If the minimal requirements are met, the resultant sequence (3'UTR Sequence Tag - UST) is aligned to the C.elegans genome using both BLAT and BLAST algorithms. The Alignment with the best score is then validated by individual curators, who assign them a unique ID number.
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Website
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Website.
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The website runs on an Apache server and is composed by a MySQL database queried by a collection of Perl CGI scripts that extract data imported from AceDB, WormBase WS180 and PicTar. So far we implemented the database to contain a comprehensive collection of all the ~26,000 transcripts described in WormBase. Where available, we provide 3'UTR annotation and evidence from ESTs and mRNA extracted from the WormBase, and prediction for putative miRNA binding sites extracted from PicTar and miRANDA. In addition, as part of the ModEncode Consortium, we integrated all the data extracted from our cloning pipeline such as ABI Trace Files, BLAT and BLAST Output, Agarose Gel evidence, Plate coordinates and Predicted folding structures. We continuously deposit into the database the cloning data from our pipeline, both raw data and our preliminary annotation. The ABI trace file applet has been adapted from Dr. Sachidanandam in Cold Spring Harbor. The 3'UTR Secondary Structure has been modeled using the mFOLD algorithm developed by Dr. Zucker in Rensselaer Polytechnic Institute.
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