Dataset RP_cDNA

General Information
Name	RP_cDNA	Species	D. melanogaster
Dataset type	cDNA sequence	FlyBase ID	FBlc0000049
Source & Content
Consists of
Created by	Berkeley Drosophila Genome Project modENCODE: Drosophila transcriptome
Available from
Strain	iso-1 (Hoskins et al., 2011)
Stage & tissue
Stage Tissue/Position (including subcellular localization) Reference embryonic stage organism embryonic stage 1 -- 17 (Hoskins et al., 2011) Comment:0-24 hr AEL
Cell Line
Recent Updates
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FB2013_03
FB2013_02
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Description & Members
Description
Parent collections	mE_Transcription_Start_Sites
Component collection(s)
Number in collection
Comment on number in collection
Members
Experimental protocol
Vector
Sample preparation
Collection preparation	Total RNA isolated from mixed stage embryos of the 'iso-1' isogenic strain was provided by the laboratory of Peter Cherbas (University of Indiana, Bloomington). Gene-specific 5' RLM-RACE products were generated using the FirstChoice RLM-RACE procedure (Ambion). Normalized pools of RACE products were created and either cloned and sequenced with an ABI3730 or directly sequenced on a 454 Life Science sequencer. Vector sequences were removed and the RNA adapter sequence was used to determine the orientation of the clone and was removed from the sequence. Each sequence represents a potential transcription start site and is oriented along the direction of transcription. Primers were designed to target all FlyBase release 5.12 (October 2008) transcript models that overlap 5' ESTs from the RE (FBrf0152058) and LD (FBrf0127297) cDNA libraries, both constructed from mixed-stage embryos. Transcripts of genes expressed in the embryo based on whole-mount RNA in situ hybridization (FBrf0205271) and literature surveys were also targeted. In all, 8570 distinct primer pairs representing 7742 genes were designed. (Hoskins et al., 2011) Nested RACE PCR reactions were carried out using HotFirePol. For 1453 RACE reactions that lacked detectable product, second-round PCR conditions were used that including 5 extra amplification cycles. Individual PCR products were pooled to create a molar-normalized mixtures of 1,440 to 2,677 products using a Tecan Genesis 2 robot. The sample pools were then concentrated to approximatley 50 ng/ul. (Hoskins et al., 2011)
Mode of assay	cDNA was characterized by high-throughput sequencing. (Hoskins et al., 2011)
Assay platform	[[Roche Genome Sequencer 454 GS FLX::www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL9333]] (Hoskins et al., 2011)
Data analysis	Clustering of RLM-RACE reads: the unique locations of all reads associated with a given target were collected, and the Euclidian distances between all pairs of distinct start locations were calculated. An iterative hierarchical clustering approach using the "complete" agglomeration method was applied to find the minimum number of clusters for which the number of TSS in a cluster is greater than three and the cluster span is less than 300 bp. To remove outliers, tags that mapped farther away than 1.5 times the inter-quartile range were excluded from a promoter region. (Hoskins et al., 2011) From 2.1 million raw RACE reads, 1.2 million were oriented, trimmed, mapped to the genome, and associated with a transcript. In total, 8418 transcripts of 7546 genes were identified. (Hoskins et al., 2011) Mapping of RLM-RACE sequences: RACE reads were processed to trim low-quality regions and adapter sequences; only sequences with a significant match to the 3’ end of the 3’-most 38 bases of the adapter sequence were retained. Only sequences that were aligned to the genome beginning at the most 5’ nucleotide were used in subsequent analyses. A read was associated with a particular transcript and RACE reaction by searching for interrogated transcripts on the same strand as the aligned sequence and within 5 kb of the 3' end of the aligned sequence. If there were multiple targeted transcripts within the region, then the sequence was assigned to the transcript and RACE reaction with the highest scoring BLAST hit to the inner transcript-specific PCR primer if applicable, or to the highest scoring BLAST hit to a targeted transcript. Each read was presumed to represent a transcription initiation event, but because the protocol relies on PCR amplification, each read does not necessarily represent an independent event. (Hoskins et al., 2011)
Additional data
	More information is available under: modENCODE: Drosophila transcriptome modMine: modENCODE_1040 modMine: modENCODE_1041 modMine: modENCODE_1042 modMine: modENCODE_1043 modMine: modENCODE_1044
Associated files
Additional sites
Comments
	Clones were not preserved and are not available for distribution.
Synonyms & Secondary IDs
Reported As
Symbol Synonym	5' RLM-RACE (Hoskins et al., 2011, Hoskins et al., 2011) RP RP_cDNA
Secondary FlyBase IDs

References ( 2 )
Research paper	Hoskins et al., 2011, Genome Res. 21(2): 182--192 Genome-wide analysis of promoter architecture in Drosophila melanogaster. [FBrf0213090]
Supplementary material	Hoskins et al., 2011, Genome Res. 21(2): Supplemental Material. [FBrf0213251]