Preparation of cDNA for Illumina Sequencing

Preparation of cDNA followed the procedure described in Mortazavi et al.^{2 (link)}, with minor modifications as described below. Prior to fragmentation, a 7 uL aliquot (∼ 500 pgs total mass) containing known concentrations of 7 “spiked in” control transcripts from A. thaliana and the lambda phage genome were added to a 100 ng aliquot of mRNA from each time point. This mixture was then fragmented to an average length of 200 nts by metal ion/heat catalyzed hydrolysis. The hydrolysis was performed in a 25 uL volume at 94°C for 90 seconds. The 5X hydrolyis buffer components are: 200 mM Tris acetate, pH 8.2, 500 mM potassium acetate and 150 mM magnesium acetate. After removal of hydrolysis ions by G50 Sephadex filtration (USA Scientific catalog # 1415-1602), the fragmented mRNA was random primed with hexamers and reverse-transcribed using the Super Script II cDNA synthesis kit (Invitrogen catalog # 11917010). After second strand synthesis, the cDNA went through end-repair and ligation reactions according to the Illumina ChIP-Seq genomic DNA preparation kit protocol (Illumina catalog # IP102-1001), using the paired end adapters and amplification primers (Illumina Catalog # PE102-1004). Ligation of the adapters adds 94 bases to the length of the cDNA molecules.