title: GSM7868188: Substantia nigra, GFP-overexpressed, rep3; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from the SN of 3-month-old mice, indicating a month after AAV administration, using TRIzol reagent (Life Technologies, #15596026). The isolated RNA was further purified using the miRNeasy Mini kit (217004, Qiagen Hilden, Germany) following the manufacturer's instructions. The concentration of total RNA was determined using the Quant-IT RiboGreen assay (Invitrogen, #R11490). To evaluate the integrity of the RNA samples, an analysis was performed using the TapeStation RNA screentape (Agilent, #5067-5576). Only high-quality RNA samples with an RNA integrity number (RIN) greater than 7.0 were selected for subsequent RNA library construction. A library was prepared independently using 1ug of total RNA for each sample by Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc., San Diego, CA, #RS-122-2101). Briefly, mRNA containing poly‐A tails was purified and collected molecules using poly‐T‐attached magnetic beads. The mRNA was then fragmented into small pieces using divalent cations under elevated temperature. The resulting fragmented RNA was reverse transcribed into first-strand cDNA using SuperScript II reverse transcriptase (Invitrogen, #18064014) and random primers. Subsequently, second-strand cDNA synthesis was performed using DNA Polymerase I, RNase H, and dUTP. These cDNA fragments underwent end repair, followed by the addition of a single 'A' base and ligation of adapters. The products are then purified and enriched with PCR to create the final cDNA library. The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (KAPA BIOSYSTEMS, #KK4854) and qualified using the TapeStation D1000 ScreenTape (Agilent Technologies, # 5067-5582). The indexed libraries were then submitted for paired-end (2x100 bp) sequencing on an Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) at Macrogen Incorporated (Seoul, Korea).