RNA Seq of plasma of patients undergoing EUS FNA procedure
Patient plasma was processed from whole blood by centrifugation twice; first at 1,000g for 10 minutes, then the supernatant plasma was removed and spun at 2,500g for 10 minutes at room temperature. Plasma aliquots were frozen and stored at -80C until we began RNA processing, which took place in batches of 8 in a randomized order for each cohort. We purified RNA from these plasma samples using a Plasma/Serum RNA purification kit available commercially from Norgen Biotek (ref. 55000). Genomic DNA was removed from the resulting RNA with Lucigen Baseline-ZERO Dnase I (ref. DB0715K) in a 20 minute incubation. The RNA was then further purified using a commercially available RNA Clean and Concentrator kit from Zymo Research (ref. R1016), and aliquoted to be stored at -80 C. We prepared sequencing libraries from 5l purified RNA for each sample using the Takara SMARTer Stranded Total RNA-seq kit v2- Pico Input Mammalian formulation (ref. 634413) according to the manufacturers instructions. We used option 2 during the library processing, without fragmentation. During the adapter addition step, we used the Takara SMARTer RNA Unique Dual Index kit -96U (ref. 634452), to append a unique index to each 5` and 3` read to aid in distinguishing samples. The final PCR amplification of the RNA-seq libraries used 16 cycles, and libraries were eluted into 20l Tris buffer and stored at -20C before sequencing.