Total RNA was isolated from cells and tissues using TRIzolreagent (cat no. 15596018, Thermo Fisher, Waltham, MA). The Stranded mRNA-Seq was performed using the Illumina NovaSeq 6000 Sequencing Systemat the University of Kansas Medical Center –Genomics Core (Kansas City, KS). Quality control on RNA submissions was completed using the Agilent TapeStation 4200 using the RNA ScreenTape Assay kit (Agilent Technologies 5067-5576). Total RNA (1ug) was used toinitiatethe library preparation protocol.The total RNA fraction was processed by oligo dT bead capture of mRNA, fragmentation, reverse transcriptioninto cDNA, end repair of cDNA, ligation withthe appropriate Unique Dual Index (UDI) adaptors, strand selection and library amplification by PCR using the Universal Plus mRNA-seq with NuQuant library preparation kit (Tecan Genomics 0520-A01).Library validation was performed using the D1000 ScreenTape Assay kit (Agilent Technologies 5067-5582) on theAgilent TapeStation 4200. Concentration of each library was determined with the NuQuant module of the library prep kit using a Qubit 4 Fluorometer (ThermoFisher/Invitrogen),Libraries were pooled based on equal molar amounts and the multiplexed pool was quantitated, in triplicate, using the Roche Lightcycler96 withFastStart Essential DNA Green Master (Roche 06402712001) and KAPA Library Quant (Illumina) DNA Standards 1-6 (KAPA Biosystems KK4903). Using the qPCR results, theRNA-Seq library pool was adjusted to 2.125nM for multiplexed sequencing.Pooled libraries were denatured with 0.2N NaOH (0.04N final concentration) and neutralized with 400mM Tris-HCl pH 8.0. A dilution of the pooled libraries to 425 pM is performed in the sample tube, on instrument, followed by onboard clonal clustering of the patterned flow cell using the NovaSeq 6000 S1 Reagent Kit (200 cycle) (Illumina 20012864). A 2x101 cycle sequencing profile with dual index reads is completed using the following sequence profile: Read 1 – 101 cycles x Index Read 1 – 8 cycles x Index Read 2 – 8 cycles x Read 2 – 101 cycles. Following collection, sequence data is converted from .bcl file format to fastq file format using bcl2fastq software and de-multiplexed into individual sequences for data distribution using a secure FTP site or Illumina BaseSpace for further downstream analysis.