sham_S1
To isolate RNA, tissue was homogenized in TRI-based reagent on ice and processed further according to the instructions on the Zymo Direct zol RNA Miniprep kit (R2051A). Phase separation was used for isolating pure RNA samples, which were then eluted in DNAse/RNAse-free water. It was later quantified for purity and concentration using a NanoDrop spectrophotometer. The prepared samples were then stored at -80 degrees Celsius for further analysis.In accordance with the instructions of Oxford Nanopore "Rapid Sequencing DNA V14 - barcoding protocol" [39], isolated total RNA per sample was reverse-transcribed with cDNA-RT adaptor and Annealing buffer at 60 degrees Celsius for 5 min, followed by ligation with NEBNext Buffer (NEB, B6058) and T4 DNA Ligase (NEB, cat# M0202M). After exonuclease treatment, samples were purified using RNAClean XP beads (Beckman Coulter, cat# A63987). Strand-switching was initiated with RT-Primer, dNTPs, and Strand-switching primer II (SQK-RBK114.24). This was followed by the addition of Maxima H minus Reverse Transcriptase (ThermoFisher, cat# EP0751) and incubation of the reaction in a thermal cycler using defined thermal cycling conditions.Next, reverse-transcribed cDNA (5 microliters) was amplified using LongAmp Hot Start Taq (NEB, M0533S) with barcode primers (SQK-RBK114.24) under optimal cycling conditions. Unbound primers were degraded with Exonuclease I (NEB, cat# M0293S) for 20 minutes. Samples were purified using AMPure XP beads (Beckman Coulter, cat# A63881), washed with 70 percent ethanol, and resuspended in Elution Buffer for downstream analysis.Equimolar cDNA quantities were then taken per sample, pooled into a library, and loaded into the flow cell FAZ07279 in the Minion Mk1b device according to the instructions from the kit SQK-RBK114.24. Base calling and demultiplexing of reads were performed with Oxford Nanopore built-in software MinKNOW (Min24.06.5).Raw sequencing reads that passed quality control (Qscore greater than 6) were aligned on the reference genome (GRCm38) [40] using the minimap2 alignment tool [41]. Gene expression quantification was performed using NanoCount [42] and normalization and differential expression analysis were conducted using the DeSeq2 package [43]. Thresholds for differentially expressed genes were set to p-value less than 0.01 and fold-change of plus or minus 15 percent.