characteristics.text: SAMN00017217
characteristics.characteristic: External Id
characteristics.text: Duke University
characteristics.characteristic: INSDC center name
characteristics.text: 2010-04-20T09:29:14.553Z
characteristics.characteristic: INSDC first public
characteristics.text: 2013-06-12T08:54:51.880Z
characteristics.characteristic: INSDC last update
characteristics.text: SRS058861
characteristics.characteristic: INSDC secondary accession
characteristics.text: live
characteristics.characteristic: INSDC status
characteristics.text: Generic
characteristics.characteristic: NCBI submission model
characteristics.text: Generic.1.0
characteristics.characteristic: NCBI submission package
characteristics.text: SRS058861
characteristics.characteristic: SRA accession
characteristics.text: Mixed stage fly embryos (0-24h) were collected according to a standard protocol. We used TRIzol reagent (Invitrogen) to extract total RNA. RNeasy Mini kit (QIAGEN) was used for cleanup and on-column DNase I digestion to remove genomic DNA according to the manufacturer's protocol. 150 μg purified RNA was enriched for poly(A)+ RNA with Dynabeads Oligo(dT)25 (Invitrogen) according to a modified protocol. 2 μg of poly(A)+ RNA was BAP/TAP (Bacterial Alkaline Phosphatase/Tobacco Acid Pyrophosphatase) treated and a chimeric linker tagged with a MmeI site was ligated to its 5' end. The RNeasy MinElute kit (QIAGEN) was used to remove excessive chimeric linkers. Random primers tagged with a MmeI recognition site were used to initiate reverse transcription. First strand cDNAs were amplified using 5 cycles of PCR, and the products were purified with DNA clean &amp;amp; concentrator-5 kit (ZYMO). Circularization was performed with a collector oligonucleotide, which converts the PCR product into a single-stranded circular DNA. After Exo I (NEB) and Exo III (NEB) digestion to remove linear DNAs, rolling circle amplification (RCA) was performed to amplify the remaining circular DNAs. The RCA products were digested with MmeI (NEB) to generate a specific 93~95bp band. The desired product was ligated with two Illumina Paired-End adaptors and amplified with low-cycle PCR. After size-selection and validation by Sanger sequencing, the final library was sequenced using an Illumina GAII with a paired-end module.
characteristics.characteristic: description
characteristics.text: Drosophila melanogaster
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_7227
characteristics.characteristic: organism
characteristics.text: Drosophila melanogaster 0-24h mixed stage embryos
characteristics.characteristic: title