AP_IsoSeq_CAST
1.5 g  mRNA isolated from > 106 was reverse transcribed with an oligo-dT20 primer with an adapter  sequence (GACTCGAGTCGACATCG-T20) using the SuperScript III First Strand Synthesis Kit  (Qiagen). 5 l of the resulting cDNA was amplified with Hot Start Phusion  Polymerase (Thermo) using primers to the adapter and the 5-UTR  (CGGCGACGTTTAAAGCTGA). 3-5 replicate PCR reactions were combined, purified  with the QIAquick PCR Purification Kit (QIAGEN), and submitted to the Johns  Hopkins Deep Sequencing & Microarray Core Facility for sequencing.  Products were size selected for the anticipated size range of 1-3kb. 1 SMRT  cell was sequenced per sample. Sequencing reads were processed in the SMRT  Analysis v4.0 software, aligned to chromosome 14 with HISAT2