title: GSM6890795: CVS_K08_ST3D_rep1; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated using TRIzol/chloroform precipitation (15596018, Thermo Fisher Scientific) as previously reported (Varberg et al., 2021). Stranded mRNA-sequencing was performed on the Illumina NovaSeq 6000 Sequencing System in the Genomics Core at the University of Kansas Medical Center. Quality control was completed with the RNA Screen Tape Assay kit (5067-5576, Agilent Technologies) on the Agilent TapeStation 4200. Total RNA (1 μg) was processed in the following steps: i) oligo dT bead capture of mRNA, ii) fragmentation, iii) reverse transcription, iv) cDNA end repair, v) Unique Dual Index (UDI) adaptor ligation, vi) strand selection, and vii) library amplification using the Universal Plus mRNA-Seq with NuQuant library preparation kit (0520-A01, Tecan Genomics). Library validation was performed with the D1000 Screen Tape Assay kit (5067-5582, Agilent) on the Agilent Tape Station 4200. Library concentrations were determined with the NuQuant module using a Qubit 4 Fluorometer (Thermo Fisher Scientific). Libraries were pooled based on equal molar amounts and the multiplexed pool was quantified, in triplicate, using the Roche Lightcycler96 with FastStart Essential DNA Green Master (06402712001, Roche) and KAPA Library Quant (Illumina) DNA Standards 1-6 (KK4903, KAPA Biosystems). Using the qPCR results, the RNA-Seq library pool was adjusted to 2.125 nM for multiplexed sequencing. Pooled libraries were denatured with 0.2 N NaOH (0.04N final concentration), neutralized with 400 mM Tris-HCl, pH 8.0, and diluted to 425 pM. Onboard clonal clustering of the patterned flow cell was performed using the NovaSeq 6000 S1 Reagent Kit (200 cycle, 20012864, Illumina). A 2x101 cycle sequencing profile with dual index reads was 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.