LOCUS AIAAD0000000 RNA linear HUM 17-APR-2009 DEFINITION Homo sapiens RNA, male 1 year infant peripheral blood monocyte TIB-202 THP-1 untreated cells target length: 42-52bp adaptar: shortRNA_3'adaptor_no_mix short rna library name: S04-CA, RIKEN fractionated small RNA library. ACCESSION AIAAD0000000 VERSION AIAAD0000000.1 KEYWORDS MGA; small RNA. 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., Hori,F., Kawazu,C., Murata,M., Nishiyori,H., Sano,H., Tagami,M. and Hayashizaki,Y. TITLE Direct Submission JOURNAL Submitted (04-DEC-2008) 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://genome.gsc.riken.jp/ REFERENCE 2 AUTHORS Taft,R.J., Glazov,E.A., Cloonan,N., Simons,C., Stephen,S., Faulkner,G., Lassmann,T., Forrest,A.R.R., Grimmond,S.M., Schroder,K., Irvine,K., Arakawa,T., Nakamura,M., Kubosak,A., Hayashida,K., Kawazu,C., Murata,M., Nishiyori,H., Fukuda,S., Kawai,J., Daub,C.O., Hume,D.A., Suzuki,H., Orlando,V., Carninci,P., Hayashizaki,Y. and Mattick,J. TITLE Tiny RNAs associated with transcription start sites in animals JOURNAL Nat. Genet., 10.1038/ng.312 (2009) In press REMARK Publication_Status: Available-Online COMMENT 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' tcg cac aga att cgc AAA-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)-UUU cag aat tcc cac cag cga gtc-(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)-gac tcg ctg gtg gga att ctg aaa-3'; Primer 2 (shortRNA5'PCRprimer): 5'(biotin)-cag acc tcg cac aga att cgc aaa-3'. The PCR products were purified and fractionated on a polyacrylamide gel. The each broad bands (corresponding to RNA of original size S01 12-22, S02 22-32, S03 32-42 S04 42-52 S05 52-82 ) 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 TAE polyacrylamide electrophoresis. After the elution from gel the RNAs were quantified, concatenated for O/N at 15 degrees Celsius with T4-DNA ligase was carried out in 5 micro l under standard conditions. Using a series of standard molecular biology techniques, short adaptors (A and B) - specific for both the 3' and 5' ends - were added to each fragment, and purify with GFX column. The libraries were sequenced by 454 Genome Sequencer 20 sequencer and then, with in in-house algorithms, the short RNA tags sequences were extracted. The algorithm searches for the linker sequences and extracts the tag if it matches the target length. THP-1 cells which were stimulated by LPS(Lipopolysaccharide) 96 hours after treatment by PMA(phorbol myristate acetate). Warning: We believe the PMA concentration may have been too high as the response of known LPS responsive genes was relatively weak, and some appeared to have been pre induced by the PMA. The Requirement for lower doses of PMA to detect LPS responses is discussed in a recent paper (E. K. Park, H. S. Jung, H. I. Yang, M. C. Yoo, C. Kim, K. S. Kim Optimized THP-1 differentiation is required for the detection of responses to weak stimuli Infl amm. res. 56 (2007) 45-50 http://www.springerlink.com/content/ f45q402760742307/fulltext.pdf). Please be aware of this when you use those data. FEATURES Location/Qualifiers source /cell_line="THP-1" /cell_type="monocyte" /clone_lib="RIKEN fractionated small RNA library" /culture_collection="ATCC:TIB-202" /db_xref="taxon:9606" /dev_stage="1 year infant" /mol_type="transcribed RNA" /note="RIKEN rna sample id : 2175" /note="adapter_name: shortRNA_3'adaptor_no_mix" /note="target length: 42-52bp" /note="create date of rna sample : 2006/07/26" /note="ligation date of short rna sequence : 2006/08/15" /note="tag production date of short rna sequence : 2006/08/18" /note="completion date of short rna library : 2006/11/06" /note="short rna library name: S04-CA" /note="experiment_condition: untreated cells" /note="time_cource: 2000 1 1 0:00" /note="comment: experimental_name:RIKEN_cell_2nd, rna_batch_name:LOT-No.3" /organism="Homo sapiens" /sex="male" /tissue_type="peripheral blood" MGA AIAAD0000001-AIAAD0012203 total number of count : 40274 Header Format >[ACC#]|[submitter's identifier]|[number of sequence count]|[map]|[free text]|[db_xref1(,db_xref2,...)]| //