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== Final Library Preparation == *Beads purified twice at (0.8x), re-suspended in 10ul *PCR on 0.5ul each library using ILMN_PCR_F and either ERCC130R227 or ERCC130R964. [[File:01082014_ERCC130_R227.jpg|400px]][[File:01082014_ERCC130_R964b.jpg|400px]] Results: *Still variable amplification levels seen with 10pg. More consistency might be achieved by combining 10pg samples for beads purification prior to TSO/PCR *Can see at least one of the 2min or 4 min showing similar levels as the 1ng (especially for the R964 - i.e.3'end) while longer PAP times tended to show poorer amplification curves... therefore, 4 min PAP may be sufficient for 10pg RNA quantities *Its difficult to fully conclude optimal conditions due to the variability... hopefully this is an artifact of the single 10pg beads purification rather than inconsistency/inefficiency of the totoRNAseq reactions at these minimal RNA concentrations
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