Targeted sequencing  of full-length TIN2 transcripts
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