Next gen sequencing nextseq platform

The procedure was performed using the Lexogen SLAM-Seq Metabolic Labeling kit. Cells were plated at 5x10⁵ cells/mL in the appropriate culture medium and were pre-treated either with 0.1% DMSO or with dTAG-VHL (500nM) for 15, 30, 60, or 120 minutes. After the appropriate pretreatment, cells were incubated with 4SU (Lexogen) at a final concentration of 100μM and were incubated with continuous dTAG-VHL/DMSO treatment for 60 min at 37°C / 5% CO₂. Upon addition of 4SU, cells were kept away from light. Cells were harvested at 300 g for 5 min at 4°C, washed with PBS and the RNA was extracted from the cell pellet using QIAshredders (Qiagen) and RNeasy Plus Mini Kit (Qiagen) according to the manufacturer’s protocol. 5ug of the extracted RNA was lodoacetamide treated according to the manufacturer’s instructions (Lexogen). Libraries were prepared using 500ng RNA and the QuantSeq 3’ mRNA-Seq Library Preparation kit (Lexogen). Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina) with 20-30 million 75bp, single-end reads.

For RNA-seq experiments, the cells were plated at 1x10⁵ cells/mL, treated with drug or 0.1% DMSO for the indicated time. Cells were sub-cultured to the original density after 3 days in fresh media with the same concentration of drug or DMSO. RNA was isolated as described above. RNA quality for RNA sequencing was checked on the Agilent TapeStation (Agilent) and quantified by Qubit (ThermoFisher). RNA (1μg) was used to make Illumina compatible libraries by doing Poly-A tail selection and library preparation using the NEBNext Ultra™ RNA Library Prep Kit for Illumina (New England Biolabs). Sequencing was done using the Illumina Next Gen Sequencing NextSeq platform (Illumina) with 20-30 million 37bp, paired-end reads.