GSM4223710: AsPC1_sgBACH1_rep3; Homo sapiens; RNA-Seq
The cells were harvested by centrifugation, and the total RNA was isolated using a RNeasy Plus Mini kit (QIAGEN) following the instruction manual. Five microgram of total RNA was treated using a GeneRead rRNA Depletion kit (Qiagen) for remove Ribosomal RNA (rRNA) and an RNeasy MiniElute kit (Qiagen) for cleanup. One hundred ng of rRNA-depleted RNA was incubated at 95 ºC for 10 min for fragmentation and was purified by Magnetic Beads Cleanup Module (Life Technologies). The libraries were constructed using Ion Total RNA-seq kit for AB Libraly builer system (Thermo Fisher Scientific Inc.) and were barcoded with Ion Xpress RNA-seq BC primer ((Thermo Fisher Scientific Inc., 16 PCR cycles). Barcoded library fragment with the size range of 100-200 bp was selected with Agencourt AMPure XP beads (Beckman Coulter, Brea, CA). Templates were prepared using Ion PI Hi-Q Chef Kit (Thermo Fisher Scientific Inc.) on Ion Chef (Thermo Fisher Scientific Inc.). Sequencing was performed on an Ion Proton system by the P1 chip v3 (Thermo Fisher Scientific Inc.) using Hi-Q Sequensing kit (Thermo Fisher Scientific Inc.).