Comparison of typical DeepSAGE and RNA-seq data generation steps.
A) DeepSAGE data preparation consists of the following basic steps: after RNA extraction the polyadenylated mRNA fraction is captured with oligo-dT beads. While RNA is still bound to the beads, double-stranded cDNA synthesis is performed. Next, cDNA is digested by NlaIII restriction enzyme (an anchoring enzyme), which cuts the DNA at CATG recognition sequences, leaving only the fragment with the most distal (3′) CATG site associated with the beads. Subsequently, a GEX adapter is attached to the 5′ end. This adapter contains a recognition sequence for the MmeI restriction enzyme that cuts the sequence 17 bp downstream of CATG site. After ligation of a second GEX adapter, fragments containing 21 bp tags (17 unknown nucleotides + CATG) are ready for sequencing. B) A typical protocol for RNA-seq data preparation has the following steps: after RNA extraction the polyadenylated mRNA fraction is captured with oligo-dT beads. Captured RNA is fragmented and for each fragment cDNA synthesis is performed using random hexamer primers. Sequencing adapters are then ligated to each fragment. This is followed by size selection of the DNA fragments and PCR amplification. Then one end of the fragment is sequenced (single-end sequencing) or both ends (paired-end sequencing).