Nucleospin rna plus xs kit

Total RNA was extracted using NucleoSpin RNA Plus XS kit, and 1 μg total RNA was used for cDNA synthesis by PrimeScript RT-PCR kit (TaKaRa, Dalian, China). The mRNA levels were examined using SYBR Green Supermix kit (Bio-Rad). All these procedures were performed according to the manufacturer's instructions. The qPCR was performed using the CFX96 Touch Real-Time PCR Detection System under conditions: 30 seconds initial denaturation at 95°C, then 40 cycles of 10 seconds at 95°C, and 30 seconds at 60°C. AKAP-9 mRNA levels were normalized to GAPDH levels. The primers were adopted from previous study [24 (link)] and listed below.
AKAP9-forward: 5′-ACTCAAGGCACAGCATAAACAC-3′
AKAP9-reverse: 5′-GTTCTTCACTGCGTC CCAA-3′
GAPDH-forward: 5′-ACAGTCAGCCGCATCTTCTT-3′
GAPDH-reverse: 5′-GACAAGCTT CCCGTTCTCAG-3′

Total RNA was isolated from each retina and processed separately using a NucleoSpin RNA Plus XS kit (TaKaRa, Tokyo, Japan), and a library was subsequently prepared using a Switching Mechanism At the 5’ end of RNA Template (SMART) method. Purified amplicon was sequenced using a Novaseq 6000 sequencer (Illumina K.K. San Diego, CA). Raw bead counts were obtained and processed using a web portal for integrated differential expression and pathway analysis (iDEP.95/iDEP1.0; http://bioinformatics.sdstate.edu/, accessed on 24 February 2023) (Ge et al., 2018 (link)). k-Means clustering was performed on the top 2000 genes ranked by standard deviation into groups based on their expression pattern across all samples. A heatmap was subsequently generated in which numerical values of points were represented by a range of colors. Identification of differentially expressed genes (DEGs) was performed using a DESeq2 algorithm by extraction with an FDR cutoff of 0.1 and min-fold change of 2 as the default setting. Pathway analysis was performed using Parametric Gene Set Enrichment Analysis (PGSEA) with gene sets from the Gene Ontology Molecular Function and Pathway and the Kyoto Encyclopedia of Genes and Genomes (KEGG). The cutoff for significance was set at 0.2.

RNA was isolated from cell pellets using Nucleospin RNA Plus XS kit (Takara), and first strand cDNA synthesis was performed using Superscript IV VILO Master Mix with ezDNAse (Thermo Fisher) per manufacturer’s instructions. PCR was performed using primers J3-RT-F2 and IGHG1-Sec-RT-R2 (secreted H chain isoforms) or IGHG1-MemEx-RT-R1 (membrane anchored H chain isoforms) (Supplementary Table 5), using 5 μL of cDNA. Resulting DNA was visualized on a 1% agarose gel with GelRed Nucleic Acid Stain (Biotium).