fc1103-lane3-ATGTCA
Illumina RNA-Seq library preparation used 2 ug total RNA and followed the standard TruSeq protocol (Part #15026498 Rev. A), modified for strand-specific sequencing by the dUTP method (Parkhomchuk et al., Nucleic Acids Res 37(18):e123, 2009). The product of first-strand cDNA synthesis is desalted to remove unincorporated dNTPs using Sephadex G-50, and the second strand synthesis used a dUTP/dNTP mixture (Fermentas #R0251) in place of the standard dNTPs to incorporate dUTP into the second strand. All subsequent steps follow the Illumina protocol, except that the PCR cocktail is prepared with an additional uracil-specific excision reagent (New England Biolabs #M5505S), and the PCR cocktail was heated to 37uC for 15 minutes prior to PCR to degrade the uracil-containing strand.