GSM7844553: NL4.3 SupT1 5dpi; Human immunodeficiency virus 1; OTHER
Total RNA was isolated using the Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA reactions were performed according to the manufacturer's instructions of the PrimeScript™ RT Reagent Kit (Takara) using primers specifically targeting the U6 and scaffold region (forward primer 5´-CCGACTCGGTGCCACTTTTT-3´, reverse primer 5´-CGTGACGTAGAAAGTAATAATTT-CTTGGG-3´). cDNA reactions were purified using the Monarch PCR Purification Kit (NEB) and eluted in 10µl elution buffer. The gRNA cassette was amplified using the NEBNext® High-Fidelity 2X PCR Master Mix and primers including Illumina adaptors and 8nt barcodes to allow Next Generation Sequencing (NGS) analysis. PCR reactions were purified using the Monarch PCR Purification Kit (NEB) and eluted in 10µl elution buffer.