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.