cDNA Capture Using IDT Xgen® Lockdown® Probes and Single-Molecule Isoform-Sequencing

One microgram of total RNA per reaction was reverse transcribed using the Clontech SMARTer cDNA synthesis kit and six sample-specific barcoded oligos dT (16mer barcode sequences, Table 1). Three reverse transcription (RT) reactions were processed in parallel for each sample. PCR optimization was used to determine the optimal amplification cycle number for the downstream large-scale PCR reactions. A single primer (primer IIA from the Clontech SMARTer kit 5′ AAG CAG TGG TAT CAA CGC AGA GTA C 3′) was used for all PCR reactions post-RT. Large-scale PCR products were purified separately with 1X AMPure PB beads, and the bioanalyzer was used for QC. An equimolar pool of 6-plex barcoded cDNA library (5 μg total) was input into the probe-based capture with a custom-designed ASFMR1 gene panel. Due to the low gene expression, five captures were performed with 5 ug input and 16 h incubation followed by 11 cycles of post-capture amplification. A SMRTBell library was constructed using 135 ng of captured and re-amplified cDNA. One SMRT Cell 1 M (20 h movie) was sequenced on the PacBio Sequel platform using 2.0 chemistry.