title: GSM8804396: Neuro-2a; Mus musculus; OTHER
library_construction_protocol: Neuro-2a, SH-SY5Y, HEK-293T, and PC-12 cells were cultured to logarithmic growth phase, reaching a density of 2×107 cells on the day of transfection. Transfections were performed using Xfect™ Transfection Reagent (Takara, Cat. No. 631317, China) under optimized conditions established from preliminary experiments. The purified plasmid DNA from the constructed library was transfected into the cells, and each experiment was conducted in triplicate to ensure reproducibility. Before cell transfection, the plasmid library was used to construct the input sequencing library. For the initial PCR, 100 ng of the plasmid library underwent a one-step PCR. The purified products were then subjected to two additional rounds of PCR with sample index primers. High-fidelity KAPA HiFi HotStart ReadyMix (Roche, Cat. No. KK2601, USA) was used in all PCR steps to ensure accurate amplification. The final products were prepared for paired-end sequencing on the Illumina NovaSeq 6000 platform. After transfection, total RNA was extracted using the E.Z.N.A.® Total RNA Midiprep Kit (Omega Bio-Tek, Cat. No. R6664, USA). Polyadenylated RNA was isolated using VAHTS mRNA Capture Beads (Vazyme, Cat. No. N401, China) and reverse-transcribed with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, Cat. No. R312-02, China). The resulting cDNA was treated with RNase A and purified using AMPure XP beads. Two rounds of PCR amplification were performed, with products purified using Agencourt SPRIselect Reagent (Beckman Coulter, Cat. No. B23318, USA). Three biological replicates were prepared for both input and output sequencing libraries, which were then sequenced using paired-end 150 bp reads on the Illumina NovaSeq 6000 platform.
identifiers.namespace: GEO
identifiers.id: GSM8804396_r1