BDGP 5 prime RACE

Celniker embryonic 5'RACE (Celniker project, Celniker subgroup)

General Description

To map transcription start sites (TSSs) of capped transcripts, 5' RLM (RNA Ligase Mediated)-RACE is performed. The 5' ends of uncapped RNAs are dephosphorylated; 5' ends of capped RNAs are protected. The 5' cap structure is then removed from capped RNAs, leaving a 5' hydroxyl group. An RNA oligonucleotide adapter is ligated to the 5' ends of uncapped transcripts. First-strand cDNA is synthesized using a random dodecamer primer, and the products are aliquoted into transcript-specific reactions. Two rounds of nested PCR are performed using transcript-specific primers; the number of cycles of amplification is limited in an effort to preserve the diversity of TSSs of each transcript. The products are cloned in the pCR2.1 vector, and the sequences of individual clones are determined using conventional capillary sequencing. The sequence data undergo base calling, vector trimming, RLM adapter identification, and quality trimming. Sequences for which the RLM adapter can be identified are aligned to the genome and, in cases where the aligned location is near the targeted transcript, transcription start sites are identified. The 5' RLM-RACE sequence reads are submitted to dbEST at Genbank.

Protocols

  1. Growth and isolation: Population Cages, Embryo Collection, RNA_extraction
  2. Sample preparation: RT, PCR and cloning
  3. Analysis: End-sequencing

Reagents

  1. Animals: Mixed Embryo 0-24 hr, Drosophila melanogaster, Y_cn_bw_sp
  2. Samples: Celniker/RNA:55, Celniker/RNA:83

Release Date: 2008-03-16