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pCMV-SPORT6.1 Library Method
Non-normalized and normalized libraries in pCMV-SPORT6.1 were constructed at Invitrogen Corp., Carlsbad, CA, which also prepared the vector. Briefly, the methods were as follows:Non-normalized (standard) libraries
- First strand cDNA was synthesized from polyA+mRNA using Invitrogen Superscript II RT and an oligo-dT primer containing a Not I site.
- Second strand cDNA was synthesized.
- The cDNA was "polished" with T4 polymerase, digested with Not I to create 5'-blunt/3'-Not I cDNA, then size-fractionated on a gel, purified, and ligated into the pCMV-Sport6.1 vector.
Normalized libraries
- The standard library was prepared.
- The library was hybridized using Cot-7 to Cot-25 values to reduce abundant sequences and increase the possibility of isolating rare or novel transcripts from the library.
- Because normalization was performed on pre-constructed libraries that had been rendered single-stranded, the average insert size and library complexity was maintained.
- Quality control of the normalized library was performed by comparing hybridization between standard and normalized library to confirm an average reduction of at least 10 fold for the abundant sequences.