GSM3133069: 50-50_PDAC3-5; Homo sapiens; RNA-Seq
After 72 hours of culture, cells were trypsinized and resuspended in PBS solution, and micromanipulated. PDAC cells or CAFs were then individually micromanipulated using a 10 μm transfer tip on an Eppendorf TransferMan NK2 micromanipulator, transferred into PCR tubes containing RNA protective lysis buffer (Clontech Laboratories), and flash frozen in liquid nitrogen. Amplified cDNA was synthesized from the entire cell lysate using the SMARTer Ultra Low Input RNA Kit for Sequencing - v3 kit (Clontech Laboratories). PCR amplification following second strand synthesis was run for 18 cycles. 1 ng of amplified cDNA was loaded into the Nextera XT kit. Normalization was done using the KAPA SYBR® FAST Universal qPCR Kit (Kapa Biosystems) rather than the bead-based normalization in the Nextera XT kit.