title: GSM8859565: Arabidopsis thaliana,output replicate 1; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: 2%CTAB was used to extract 3 μg of Arabidopsis thaliana Col-0 genomic DNA First, Arabidopsis thaliana genomic DNA fragmented using ultrasonication. The fragmented DNA was size-selected (450–500 bp). DNA fragments underwent end repair, adapter ligation, and purification. Adapter-ligated DNA was then amplified using PCR, incorporating homologous arms for cloning. The purified PCR products, now containing homologous arms, were inserted into the linearized vector backbone. After recombination, all products were pooled, and DNA constructs were subsequently transformed into MegaX DH10B™ T1R electrocompetent cells. All transformed Escherichia coli cultures were pooled and grown in LB medium. The input plasmid library was extracted. The input plasmid library was introduced into Agrobacterium tumefaciens strain GV3101 carrying the helper plasmid pSoup via electroporation. The transformed Agrobacterium culture was grown in YEP medium. The bacteria were harvested and resuspended in infiltration buffer. The suspension was then infiltrated into the leaves of Nicotiana benthamiana plants. The plants were maintained under normal growth conditions for 72 hours. Total RNA was extracted from the transformed Nicotiana benthamiana leaves. Polyadenylated  mRNA was then isolated. The mRNA was treated. First-strand cDNA was synthesized using SuperScript III. Target-specific reverse transcription (RT) was performed. After RT, the reaction was treated with RNase A and H, followed by pooling. In the final PCR amplification, cDNA reaction was used as a template for PCR reaction. Each index PCR was performed. The PCR products were size-selected (450–500 bp) and submitted for Illumina sequencing.The input library for sequencing was generated using a similar PCR setup.