GSM8463335: Sample_KO2; Mus musculus; RNA-Seq
RNA isolation was performed using the TRIzol-chloroform method (Thermo Fisher Scientific, Cat. No. 15596018). Bulk mRNA sequencing was conducted at the University of Kansas Medical Center – Genomics Core, utilizing the Illumina NovaSeq 6000 Sequencing System. The process began with a quality control step, where RNA samples were assessed using the TapeStation 4200 equipped with RNA ScreenTape from Agilent Technologies. Library preparation involved 500 ng of total RNA, which was processed to isolate mRNA through oligo dT bead capture. This mRNA was then fragmented and reverse transcribed into cDNA. The cDNA underwent end repair, ligation with Unique Dual Index (UDI) adaptors, strand-specific selection, and PCR amplification using the Universal Plus mRNA-seq with UDI preparation kit from Tecan Genomics. Following the preparation, library validation was conducted using DNA 1000 ScreenTape on the TapeStation 4200 to assess the  quality and size of the libraries. The concentration of each library was quantified using the NuQuant method on a Qubit 4 Fluorometer, and libraries were normalized and pooled to a concentration of 6nM. This pool was further validated and adjusted to a final concentration of 1.9nM for sequencing, using the Roche LightCycler96 with FastStart Essential DNA Green Master and KAPA Library Quant DNA Standards. For sequencing, pooled libraries were denatured, neutralized, and diluted before being loaded onto the NovaSeq 6000. A sequencing cycle of 2x101, with additional cycles for indexing, was performed using the S1 Reagent Kit. Post-sequencing, data were converted to fastq format using bcl2fastq, de-multiplexed, and made available for downstream analysis via secure FTP or Illumina BaseSpace.