title: GSM8525839: B2R.2; Rattus norvegicus; RNA-Seq
library_construction_protocol: Total RNA was extracted from cultured NPCs using the TRIzol Reagent (AG RNAex Pro Reagent, China). RNA degradation and contamination were assessed by electrophoresis on a 1% agarose gel. The quantity of RNA was measured using both the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA) and the Nanodrop One spectrophotometer (Thermo Fisher Scientific, MA, USA). RNA integrity was accurately determined using the Agilent 4200 TapeStation system (Agilent Technologies, Waldbron, Germany). Library construction was performed using the ALFA-SEQ RNA Library Prep Kit (Alfa Genetics, China). The key steps included: a) Purification of mRNA from total RNA using poly-T oligo-attached magnetic beads. b) Fragmentation of mRNA using the RT Buffer provided in the ALFA-SEQ RNA Library Prep Kit. c) First-strand cDNA synthesis using random hexamer primers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. d) Terminal repair and 3' terminal adenylation. e) Adapter ligation, fragment selection, and purification. f) PCR amplification using Hi-Fi PCR Mix and primers. g) Purification of PCR products with magnetic beads, followed by evaluation of library fragment size using the Qsep400 high-throughput nucleic acid protein analysis system (Hangzhou Houze Biotechnology Co., LTD., China). The concentration of the final library was measured using the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Waltham, USA).