LOCUS       ADAAB0000000                       RNA     linear   HUM 23-JUL-2008
DEFINITION  Homo sapiens unassigned RNA, male 15 years liver hepatoma RCB-1648
            HepG2 80bp, RIKEN fractionated small RNA library.
ACCESSION   ADAAB0000000
VERSION     ADAAB0000000.1
KEYWORDS    MGA; unspecified tag.
SOURCE      Homo sapiens
  ORGANISM  Homo sapiens
            Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
            Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;
            Catarrhini; Hominidae; Homo.
REFERENCE   1
  AUTHORS   Carninci,P., Fukuda,S., Hasegawa,A., Hayashida,K., Hori,F.,
            Nakamura,M., Nishiyori,H., Sano,H., Tagami,M. and Hayashizaki,Y.
  TITLE     Direct Submission
  JOURNAL   Submitted (13-APR-2007) to the DDBJ/EMBL/GenBank databases.
            Contact:Yoshihide Hayashizaki
            The Institute of Physical and Chemical Research (RIKEN), Omics
            Science Center, RIKEN Yokohama Institute; 1-7-22 Suehiro-cho,
            Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan
            URL    :http://www.osc.riken.jp/
REFERENCE   2  
  AUTHORS   Kawaji,H., Nakamura,M., Takahashi,Y., Sandelin,A., Katayama,S.,
            Fukuda,S., Daub,C.O., Kai,C., Kawai,J., Yasuda,J., Piero,C. and
            Hayashizaki,Y.
  TITLE     Hidden layers of human small RNAs
  JOURNAL   (er) BMC Genomics, 9:157, doi:10.1186/1471-2164-9-157 (2008)
COMMENT     small rna libraries were prepared and sequenced in Genome
            Exploration Research Group Genomics Science Center (GSC) :
            (Present) Omics Science Center in RIKEN Yokohama Institute
            
            Please visit our web site for further details.
            URL:http://www.osc.riken.jp/
            
            Total RNA was extracted by acid guanidinium phenol chloroform
            (AGPC). All precipitations were done with ethanol instead of
            isopropanol to ensure the recovery of short RNAs. The
            oligonucleotide ligation was carried out by using total RNA and a
            5' oligonucleotide RNA/DNA chimeric (5' phosphorylated, 3' RNA)
            containing an EcoRI recognition site (sequence:
            5'-acggaattcctcactAAA-3', the upper-case indicates the RNA part of
            the oligonucleotide, lower-case indicates the DNA part of the
            oligonucleotide) and a specific 3' oligonucleotide, containing
            another EcoRI recognition site (sequence:
            5'-phosphate-UUUaaccgcgaattccag-biotin-3', the upper-case
            indicates the RNA part of the oligonucleotide, lower-case
            indicates the DNA part of the oligonucleotide).
            Ligated, short RNAs were separated from 5'/3'adaptor dimers on a
            polyacrylamide denaturing gel. 37bp~200bp short RNAs, running
            above adaptor dimers, were excised and eluted from the gel in
            elution buffer. The cDNA synthesis was carried out from the
            purified short RNAs using the 3'-RT-PCR primer (sequence:
            5'-gactagctggaattcgcggttaaa-3') and MMLV reverse transcriptase
            RNaseH minus (Promega). The DNA products derived from short RNAs
            were amplified by PCR using adaptor-specific primers: Primer 1
            (shortRNA3'RT-PCRprimer): 5'-biotin-gcacgctggcctcgtgagaattc-3';
            Primer 2 (shortRNA5'PCRprimer): 5'-cagccaacggaattcctcactaaa-3'.
            The PCR products were purified and fractionated on a
            polyacrylamide gel. Two broad bands (corresponding to RNA of
            original size 20-25 and 30-40) were cut out of the gel, crushed
            and eluted from the gel. After a second cycle of PCR, the products
            were digested with EcoRI, followed by purification of the insert
            and removal of the remaining fraction corresponding to the
            digested linker, by 12% native TBE polyacrylamide electrophoresis.
            After the elution from gel the RNAs were quantified and
            concatenated for 20 min at 16 degrees Celsius with T4-DNA ligase
            under standard conditions. After purification, the concatamers
            were cloned into the pZErO-2 cloning vector, previously cleaved
            with EcoRI, and the ligation with the T4 ligase was carried out in
            5 micro l under standard conditions. After overnight ligation, the
            plasmid library was electroporated into DH-10b ultracompetent
            cells and subjected to sequencing.
            The short RNA libraries were sequenced with Sanger methods and
            then, with in in-house algorithms, the short RNA tags sequences
            were extracted. Short RNA tags were extracted with the following
            parameters: vector masking, minimum 12-bp recognition allowed;
            EcoRI ligated doublet linker (24bp) masking: maximum mismatch, 2
            bp allowed; short RNA tags length, no limits.
FEATURES             Location/Qualifiers
     source          
                     /cell_line="RCB-1648 HepG2"
                     /cell_type="hepatoma"
                     /clone_lib="RIKEN fractionated small RNA library"
                     /db_xref="taxon:9606"
                     /dev_stage="15 years"
                     /mol_type="unassigned RNA"
                     /note="carve out tag length: 80bp"
                     /note="create date of short rna library: 2005/08/17"
                     /note="short rna library name: S15-BA"
                     /note="RIKEN rna library name: HFW"
                     /organism="Homo sapiens"
                     /sex="male"
                     /strain="caucasian"
                     /tissue_type="liver"
MGA         ADAAB0000001-ADAAB0006146
            total number of count : 15342
            Header Format
            >[ACC#]|[submitter's identifier]|[number of sequence
            count]|[map]|[free text]|[db_xref1(,db_xref2,...)]|
//