mRNA isolation and sequencing
Total RNA was isolated from the brain’s hippocampus region by using the TRI reagent (sigma T9424) according to the manufacturer’s instructions. Briefly, 50–100 mg of brain tissue samples were homogenized in 1 ml of TRI reagent using a polytron homogenizer (PT 2500 E). The samples were incubated for five minutes at room temperature, after that, 0.2 ml of chloroform per ml of TRI reagent was added and vortexed and incubated at RT for 10 minutes. The samples were centrifuged at 14,000g for 15 minutes at 4 °C, the upper aqueous phase containing RNA was collected and 2-propanol (0.5 ml per 1 ml of TRI reagent) was added and incubated at RT for the next 10 minutes. Further, centrifugation was done at 12,000 g for 10 minutes at 4 °C. The supernatants were discarded, and 75% ethanol (1 ml per 1 ml of TRI reagent used) was added to the resultant RNA precipitate pellet. The samples were sent to the Neuberg Centre for Genomic Medicine, Ahmedabad, India, for further processing and sequencing. According to them, the RNA quality was assessed by Qubit™ 4 Fluorometer (Thermofisher, Q33238) using an RNA High Sensitivity (HS) assay kit (Thermofisher, Q32852) following the manufacturer’s instruction. All RNA samples with RNA Integrity Number (RIN) greater than 7.2 were used for library preparation. libraries were prepared with 25 ng of mRNA, poly(A)-selected from 5 µg total RNA, with 13 cycles of PCR amplification. Libraries were quantified by Qubit™ 4 Fluorometer using a DNA High Sensitivity assay kit (Thermofisher, cat no. Q32851) following the manufacturer’s protocol. Library size was confirmed by Tapestation 4150 (Agilent) utilizing highly sensitive D1000 screentapes (Agilent, cat no. 5067- 5582) following manufacturers’ protocol. Pooled library was loaded onto NOVASEQ 6000 high-output flow cell from Illumina (FC-404-2002), and 150 base pair pair-end sequencing was performed on a NOVASEQ 6000 sequencer. Libraries were sequenced to an average depth of 40 million reads per sample. Sequences were aligned to the M14 genome (protein coding, https://www.gencodegenes.org/mouse/release_M14.html) with STAR (v2.7.10a) and assigned to features with subread: feature Counts (v2.0.1). The raw data were processed by R statistical software (v 4.2.2, https://www.r-project.org/). The feature count data were annotated with org.Mm.eg.db (Annotation Dbi) to get the gene symbols. Gene IDs without gene annotation or with multiple gene symbols were removed. We got a total of 30951 genes after removing 55 duplicated genes.