GSM9271408: SH-SY5Y cells, ρ0, sample 4; Homo sapiens; RNA-Seq
RNA isolations from our ρ0 and ρ+ cell lines were performed using a Qiagen miRNeasy Mini kit (217004) with on column DNase treatment using a Qiagen RNase-Free DNase Set (79254). The stranded RNA-seq was performed using an Illumina NovaSeq 6000 Sequencing System at the University of Kansas Medical Center Genomics Core (Kansas City, KS). Quality control of the total RNA submissions was completed using an Agilent TapeStation 4200 using an RNA ScreenTape Assay kit (Agilent Technologies 5067-5576). Total RNA (1 ug) was used to initiate the library preparation protocol. The total RNA fraction was processed by oligo dT bead capture of mRNA, fragmentation of enriched mRNA, reverse transcription into cDNA, end repair of cDNA, ligation with the appropriate Unique Dual Index (UDI) adaptors, and strand selection and library amplification by PCR using a Universal Plus mRNA-seq with NuQuant library preparation kit (Tecan Genomics 0520-A01). Library validation was performed using a D1000 ScreenTape Assay kit (Agilent Technologies 5067-5582) on an Agilent 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 normalized to 4 nM concentration and pooled. The multiplexed pool was quantitated, in triplicate, using a Roche Lightcycler96 with FastStart Essential DNA Green Master (Roche 06402712001) and KAPA Library Quant (Illumina) DNA Standards 1-6 (KAPA Biosystems KK4903). Using the qPCR results, the RNA-Seq library pool was adjusted to 1.9 nM for multiplexed sequencing. Pooled libraries were denatured with 0.2 N NaOH (0.04 N final concentration) and neutralized with 400 mM Tris-HCl pH 8.0. A dilution of the pooled libraries to 380 pM was performed in the sample tube, on instrument, followed by onboard clonal clustering of the patterned flow cell using a NovaSeq 6000 S1 Reagent Kit v1.5 (200 cycle) (Illumina 20028318). 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.