SuperScript® hESC cDNA Libraries: Library Construction and Library Statistics

SuperScript® hESC cDNA libraries were constructed using SuperScript® III reverse transcriptase for first-strand synthesis to generate high percentages of full-length and high-yield cDNA.

To construct the BG01v cell line standard library, double strand cDNA was digested with Not1 and directionally ligated into the Gateway®-compatible library vector pCMV-SPORT6.1. The standard library was quality tested for the average insert size, the percent recombinants, and the titer (cfu/ml) of the library (Table 1).

To construct full-length normalized cDNA libraries for BG01, BG02, and BG03 hESC cell lines, first strand cDNA was enriched for full-length transcripts by selectively isolating full-length mRNA:cDNA hybrids prior to second strand synthesis. Double strand cDNA was digested with Not1 and directionally ligated into pCMV-SPORT6.1. Following ligation, the primary library was normalized to reduce the abundance of highly abundant genes (HAG) as measured by the genes EF-1alpha and Beta-tubulin. The full-length, normalized libraries were quality tested for the average insert size, the percent recombinants, the percent of full-length clones, the fold reduction of high abundant genes, and the titer (cfu/ml) of the library (Table 1).

SuperScript® hESC cDNA library statistics:

Table 1. Specification and QC data of hESC cDNA libraries

	Specification	cDNA Library Statistics
Cell line name		BG01v	BG01	BG02	BG03
Average insert size	≥ 1.2 kb	1.9 kb	1.9 kb	2.0 kb	2.1 kb
% recombinants	≥ 87%	100%	92%	100%	96%
% full-length clones	≥ 60%	n/a	91%	89%	86%
Fold reduction of high abundant genes	≥ 10 fold	n/a	16 fold	16 fold	34 fold
Titer (cfu/ml)	≥ 1x10e9	3x10e10	3.6x10e9	7x10e9	1x10e9

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