GSM8962771: 12106-YS-0009,Saline; Mus musculus; RNA-Seq
7 days after SI/saline injection, RPE was collected by removing the retina and incubating posterial eyecup in RNAprotect solution (QIAGEN). RNA was extracted using RNeasy Plus Micro Kit (QIAGEN). A Quality Control analysis was performed on the RNA samples using the Agilent Bioanalyzer to assess quality and an RNA Qubit assay was used to determine quantity.  Library preparation was completed using the NEBNext® rRNA Depletion kit and protocol (P/N: E7850X, NEB, Ipswich, MA) per manufacturer recommendations (RNA inputs ranging from 74ng-250ng). The kit employs an RNaseH-based method to deplete both cytoplasmic and mitochondrial rRNA. After ribosomal depletion, the RNA samples underwent mRNA enrichment using oligo(dT) beads, followed by thermal fragmentation using divalent cations under elevated temperature.  First-strand cDNA synthesis was performed using random hexamer primers, while second-strand cDNA synthesis was carried out using DNA Polymerase I and RNase H. End repair, A-tailing, and adapter ligation was performed to generate the final cDNA library. The library quality was assessed using a Bioanalyzer and quantified using a qPCR-based method with the KAPA Library Quantification Kit (P/N: KK4873) and the QuantStudio 12K instrument. Prepared library pools are pooled in equimolar ratios based on the qPCR results, and the resulting pool is subjected to cluster generation using the NovaSeq X Plus System, following the manufacturer's protocols. 150 bp paired-end sequencing is performed on the NovaSeq X Plus platform targeting 70M reads per sample.  Raw sequencing data (FASTQ files) obtained from the NovaSeq X Plus is subjected to quality control analysis, including read quality assessment. Real Time Analysis Software (RTA) and NovaSeq Control Software (NCS) (1.8.0; Illumina) is used for base calling. MultiQC (v1.8; Illumina) is used for data quality assessments.