GSM8197569: NK_UN_N7; Mus musculus; RNA-Seq
RNA from sorted cells was extracted using the RNeasy mini Kit as per manufacturer instructions. DNA was removed using DNA-removal columns (Quiagen). RNA quantity and integrity were determined by Agilent 5400, and only samples with > 100 ng RNA and RNA integrity number > 4 were used. mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads. The isolated mRNA was subjected to fragmentation, followed by the synthesis of first-strand cDNA employing random hexamer primers. Subsequently, second-strand cDNA synthesis was performed with dTTP to create a non-directional library. The prepared library underwent a series of steps, including end repair, poly-A enrichment, adapter ligation, size selection, amplification, and purification. The library quality and quantity were assessed using Quibit and real-time PCR for quantification and a bioanalyzer for size distribution analysis. Quantified libraries were pooled and subjected to next-generation Illumina sequencing, generating paired-end reads.