Read Counts_Bulk RNAsequencing_PFC_Naïve and Adolescent Stressed Animals

Animals: Male and female Sprague-Dawley rats (postnatal day, PND70), an outbred laboratory rat population, were obtained from the Central Animal Facility of the University of São Paulo, Ribeirão Preto and allowed to acclimatize for one week in the local animal facility before breeding. Mating was confirmed by spermatozoa presence in the vaginal smear, and birthday was defined as PND0. Pups were weaned on PND21. We use only the male offspring, as female adolescent rats do not show the behavioral and electrophysiological changes induced by this stress protocol applied from PND31–40. Animals were randomly assigned to experimental groups, each cage devoted to a specific experimental procedure. Rats were housed (2 – 3 animals per cage) at temperature- (22°C) and humidity-controlled (47%) room. The Ribeirão Preto Medical School Ethics Committee (CEUA-FMRP 248/2019) approved the procedures following Brazilian and international regulations.<br><br>Stress Protocol: Animals were exposed to inescapable foot shock (FS; from PND31 – 40) daily and three restraint stress (RS) sessions (PND31, 32, and 40). Briefly, rats were exposed to one session of FS per day for 10 consecutive days. In each session, animals were placed in a Plexiglas chamber with a grid floor of 0.48 cm stainless steel rods spaced 1.6 cm apart (EP107R, Insight Equipment, Brazil). 25 FS (1.0 mA, 2 s) was delivered pseudo-randomly (5 cycles of 30, 60, 40, 60, and 90 seconds). Immediately after receiving FS, rats were submitted to RS for 1 hour in Plexiglas cylindrical restraint tubes (20.3×5.1 cm) ventilated by holes (1 cm diameter) on the first, second, and last day of FS exposure. Naïve animals (odd numbers) were left undisturbed in their home cages (n = 8), while stressed animals (even numbers) (n = 8) were subjected to the stress protocol.<br><br>RNA isolation: On PND 51, animals were anesthetized (urethane 25%, 1 mL/100g/rat) and perfused with cold 0.01M phosphate-buffered saline (PBS, pH=7.4). PFC from both hemispheres was collected and snapped frozen in liquid nitrogen until use for RNA extraction (n=8/group). To this end, we used RNAqueous-Micro Total RNA Isolation Kit (ThermoFisher Scientific; #AM1931), according to the manufacturer's instructions. <br><br>Library strategy: bulk RNA-Seq<br>Library source: transcriptomic<br>Library selection: cDNA<br>Instrument model: Illumina HiSeq 1000<br><br>Bulk RNA-sequencing. The extracted RNA was used for performing the transcriptomic analysis from the PFC of naïve and adolescent-stressed rats using bulk RNA barcoding and sequencing. Briefly, the RNA samples were reverse transcribed with individual barcoded oligo-dT primers. Then, all samples were pooled together, and the second strand synthesis generated the double-stranded cDNA via the nick translation method. Illumina-compatible libraries were prepared by tag-mentation of 5 ng of full-length double-stranded cDNA. Then, the library was amplified, profiled, and sequenced using the Illumina NextSeq 500 platform. Transcriptomic analysis. Following a quality assessment with FastQC, gene reads were mapped with HISAT2 onto Rnor_6.0/rn6 genome assembly for Rattus norvegicus. Mapped reads were counted for each gene locus using the featureCounts function of the subread (2.0.2) package. <br><br>Code for animals in read count file: odd number = naive animals; even numbers = stressed animals