title: GSM9429437: RNA-seq, humanized mouse, brain tissue, biological replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized via carbon dioxide asphyxiation followed by cervical dislocation, in accordance with institutional animal care guidelines. Tissues were immediately harvested, briefly rinsed in ice-cold phosphate-buffered saline (PBS), and placed in RNAlater (Invitrogen) and stored at −20 °C until RNA extraction. For RNA isolation, tissues were removed from RNAlater and washed in ice cold PBS prior to homogenization in TRIzol reagent. MEFs were washed with PBS and lysed directly in plates using TRIzol for RNA extraction. Total RNA was extracted by column-based purification with the Direct-zol RNA MiniPrep Kit (Zymo Research), according to the manufacturers' instructions. Genomic DNA was removed during purification using DNaseI supplied with kit. RNA quality and integrity were assessed using an Agilent Bioanalyzer, and only samples with high RNA integrity numbers (RIN) and A260/A280 ratios between 1.8–2.1 were used for library preparation. Purified RNA was stored at −80 °C until processing. Polyadenylated mRNA was isolated from 1 µg of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) and converted into sequencing libraries using the NEBNext Ultra II RNA Library Prep Kit for Illumina, according to the manufacturer's instructions. Libraries were fragmented, reverse transcribed, end-repaired, adapter-ligated, and amplified by limited-cycle PCR. Library size distribution (200–400 bp) was confirmed using an Agilent Bioanalyzer. Sequencing was performed on an Illumina NovaSeq platform to generate paired-end reads.