PacBio and Illumina Sequencing of Full-Length cDNA

Total RNA isolation from cultured cells (two biological replicas per cell type) was done with the Nucleospin RNA kit (Macherey-Nagel) obtaining RINs (RNA Integrity Numbers) between 10 and 9.7 for all samples. The synthesis of full-length cDNA was performed with the SMARTer PCR cDNA Synthesis kit (Clontech, version 040114) following PacBio recommendations. The cDNA synthesis protocol used 1 µg of total RNA, 42°C for retrotranscription, and 13 PCR amplification cycles to control for overamplification of small fragments. For each sample, we performed two first-strand cDNA synthesis reactions and nine PCR reactions using 10 µL of first strand cDNA (diluted 1:5 in TE-buffer) to obtain ∼14–16 µg full-length cDNA per sample. Each sample was submitted to the ICBR sequencing facility (University of Florida) for PacBio sequencing (P4-C2 chemistry). Three cDNA fractions were obtained with BluePippin and sequenced on the RSII instrument using two SMRT cells for the 1–2 kb fraction, and three SMRT cells for 2–3 and 3–6 kb fractions, for a total of eight SMRT cells per sample. Additionally, the same samples were sequenced with the Illumina NextSeq instrument using Nextera tagmentation and 2×50 paired-end sequencing.