LOCUS ALAAA0000000 RNA linear ROD 15-JUN-2010 DEFINITION Mus musculus RNA, unfertilized egg small RNA. ACCESSION ALAAA0000000 VERSION ALAAA0000000.1 KEYWORDS MGA; small RNA. SOURCE Mus musculus (house mouse) ORGANISM Mus musculus Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Sciurognathi; Muroidea; Muridae; Murinae; Mus. REFERENCE 1 AUTHORS Totoki,Y., Toyoda,A. and Hohjoh,H. TITLE Direct Submission JOURNAL Submitted (22-APR-2009) to the DDBJ/EMBL/GenBank databases. Contact:Hirohiko Hohjoh National Institute of Neuroscience, Department of Molecular Genetics; Ogawahigashi 4-1-1, Kodaira, Tokyo 187-8502, Japan REFERENCE 2 AUTHORS Ohnishi,Y., Totoki,Y., Toyoda,A., Watanabe,T., Yamamoto,Y., Tokunaga,K., Sakaki,Y., Sasaki,H. and Hohjoh,H. TITLE Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development JOURNAL Nucleic Acids Res., doi:10.1093/nar/gkq229 (2010) In press REMARK Publication Status: Available-Online prior to print COMMENT Total RNA was extracted from 1470 mouse oocytes, 960 mouse embryos at the 8- to 16-cell stage, 438 blastocysts and 405 ICMs using TRIzol reagent (Invitrogen), and subjected to size fractionation via flash PAGE (Ambion) according to the manufacturer's instructions. Approximately 18- to 40-nt RNA fragments were collected and subjected to ligation with 5 uM of the 3'-adaptor RNA oligonucleotide (Linker-1), that was 5'-adenylated and 3'-blocked with a dideoxy-C base by T4 RNA ligase (Amersham) without ATP at 37 degrees C for 1 h. The RNAs were then purified by polyacrylamide gel electrophoresis, eluted from gels in elution buffer (0.5 M ammonium acetate, 1 mM EDTA and 0.1% SDS), collected by ethanol precipitation and dissolved in H2O. The collected RNAs were further ligated to the 5'-adaptor RNA oligonucleotide by T4 RNA ligase in the presence of ATP at 37 degrees C for 1 h, and used as templates to synthesize the first strand complementary DNAs (cDNAs) using SuperScript II reverse transcriptase (Invitrogen) with the 3' PCR Oligo complementary to the Linker-1 (3'-adaptor oligonucleotide) according to the manufacturer's instructions. The resultant cDNAs were subjected to amplification by PCR using the ABI GeneAmp PCR system 9700 (Applied Biosystems). The thermal cycling was carried out as follows. In the first PCR, template and 5' PCR Oligo and 3' PCR Oligo primers were heat denaturation at 96 degrees C for 1 min, followed by 20 cycles of amplification at 95 degrees C for 10 s, 50 degrees C for 1 min and 72 degrees C for 20 s. The second PCR was carried out using the first PCR product and the second and third PCR-F and -R primers for 8-10 cycles of the thermal cycling profile in the first PCR. The third PCR was carried out using the second PCR product with 8 cycles of the thermal cycling profile in the second PCR. The PCR products were purified by polyacrylamide gel electrophoresis, and collected and dissolved in TE (pH 8.0) as described above after every PCR amplification. Sequence determination of the cDNAs prepared from small RNAs was carried out using the 454 pyrosequencing technology (GS-20) (Roche). We removed linker sequences from raw reads data and extracted only 17-40 nt small RNA sequences which located between 5' linker and 3' linker. FEATURES Location/Qualifiers source /db_xref="taxon:10090" /dev_stage="metaphase II oocyte" /mol_type="transcribed RNA" /organism="Mus musculus" /strain="ICR" /tissue_type="unfertilized egg" MGA ALAAA0000001-ALAAA0130942 total number of count : 269049 Header Format >[ACC#]|[submitter's identifier]|[number of sequence count]|[map]|[free text]|[db_xref1(,db_xref2,...)]| //