GSM7091376: AAV_SYN_GFP_2h_Mg_(2) [KSZ_F83_0011]; Rattus norvegicus; RNA-Seq
RNA isolation from cell culture: To isolate RNA from the cell culture, the plate was washed twice with sterile, cold PBS buffer. After the buffer was drawn off, 400 µl of Qiazole (#79306, Qiagen) was added to the well of a plate, and incubated 5 minutes at room temperature with shaking. Next, the homogenate was pipetted several times and transferred to new tubes. Eighty µl of chloroform (#C0549-1PT, Sigma) was added to the homogenate. The samples were vortexed and incubated 10-15 minutes at room temperature until phase separation. The upper phase was used for further RNA isolation, which was performed according to the protocol of the RNA isolation kit: RNeasy Mini Kit (#74104, Qiagen). The RNA was measured on a Nanodrop Spectrophotometer (DS-11 Spectrophotometer, DeNovix) at λ= 260 nm and 280 nm. The isolated RNA was stored at -80°C. RNA-seq libraries were prepared by KAPA Stranded mRNA Sample Preparation Kit according to the manufacturer's protocol (Kapa Biosystems, MA, USA) as previously described (Grabowska et al., 2022). Transcriptomic data analysis was done as follows: FASTQ files were aligned to rn6 rat reference genome with STAR program (Dobin et al., 2013), reads were counted to genes using feature Counts algorithm (Liao et al., 2014). Gene counts were normalized with FPKM method and differential analysis was performed by DESeq2 (Love et al., 2014). Genes were considered to be differentially expressed (DE) with adjusted p value < 0.05(Yu et al., 2012).  In order to examine gene expression patterns, cluster analysis was performed in cultures after transfection with the respective viral constructs, in R software using the Mfuzz Clustering package.