5' RLM-RACE sequencing, Adults
Dm Adult 5-primeRACE 454 sequencing (Celniker project)
To map transcription start sites (TSSs) of capped transcripts, 5-prime RLM (RNA Ligase Mediated)-RACE (Rapid Amplification of cDNA Ends) is performed. The 5-prime ends of uncapped RNAs are dephosphorylated; 5-prime ends of capped RNAs are protected. The 5-prime cap structure is then removed from capped RNAs, leaving a 5-prime phosphate group. An RNA oligonucleotide adapter is ligated to the 5-prime ends of the decapped 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 sequenced by one of two methods. In the first method, the products are cloned in the vector pCR2.1, and the sequences of individual clones are determined using conventional capillary sequencing. In the second method, RACE products are analyzed on agarose gels to estimate product sizes and concentrations, and normalized equimolar pools of 1,000 to 5,000 products are sequenced on the Roche 454 Life Sciences platform. The sequence data then undergo base calling, vector or linker 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 a transcript targeted in the pool, transcription start sites are identified. The 5-prime RLM-RACE sequence reads are submitted to dbEST (capillary sequencing) or the Short Read Archive (454 sequencing) at Genbank.
- Growth and isolation: RNA processing for RACE
- Sample preparation: RT, Nested PCR, Pooling, RT
Related modENCODE submissions:
- Data Analysis: Pooling, Pooling