AAV-injected mice were subjected to extinction learning or retrieval of fear memory 24 hours after conditioning. The vHPC tissues were collected 2 hours after behavioral test and rapidly frozen in liquid nitrogen. Samples from four mice that received the same AAV injection (AAV-Syn-Cre-GFP or AAV-Syn-GFP) and behavioral tests (Ext. or No Ext.) were pulled for RNA sequencing, and for each condition, the RNA sequencing was performed on two independent sets of samples.

Total RNA was extracted using a mirVana miRNA Isolation kit (catalog no. AM1561, Ambion) following the manufacturer’s instructions. The quantification and integrity of the RNA samples obtained were determined using Qubit 2.0 Fluorometer (Life Technologies, Waltham, MA) and Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara), respectively. Only samples with RNA integrity numbers of >8 (out of 10) were used for sequencing. Samples were prepared for sequencing by Shanghai Biotechnology Corporation using the VAHTS Stranded mRNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing) and barcoded to allow samples to be multiplexed within a flow cell lane. Barcoded complementary DNA (cDNA) libraries were sequenced on a single lane of the Illumina HiSeq X Ten Sequencing System to obtain 150–base pair (bp) single-end reads at an approximate sequencing depth of 25.6 to 30.3 million reads per sample. Raw reads were trimmed to remove sequencing artifacts (1 bp from 3′ end) and filtered to remove low-quality reads (reads with quality scores of <20 in more than 10% of bases were discarded) before alignment to mouse genome (mm10) using HISAT2 (version 2.0.4). Differential expression analysis was conducted with edgeR, which was used to quantify transcript reads and to obtain z scores and fold change values for individual genes. Genes with corrected P value of ≤0.05 (Benjamini-Hochberg FDR correction) and fold change of ≥1.5 were selected for further analysis. Gene ontology categories were manually curated from results.

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