Illumina NovaSeq 6000 sequencing; RNA seq of MLL-ENL transformed cells under serine/glycine starvation including a starvation resistant clone
RNA seq of MLL-ENL transformed cells under serine/glycine starvation including a starvation resistant clone
Cells were grown in MEM supplemented with 10% FCS, pen/strep and 1x MEM essential amino acids (all ThermoFisher, Dreieich Germany) for starvation (minimal) media. For fully supplemented media serine and glycine was added to 400µM each. Cells were labeled for 1h by addition of 4-thiouridine at 100µM and harvested (approx. 10x10e6 cells)by centrifugation for lysis and nucleic extraction. cells grown in minimal medium (starvation) Nascent RNA isolation was performed exactly as described in Garcia-Cuellar et al. CellReports S2211-1247(16)30259-5. In short RNA was isolated with Qiagen RNAeasy column purification followed by biotinylation with Biotin-HPDP. Labeled RNA was isolated by Streptavidin coupled beads (µBeads) from Miltenyi (µMACS) exactly as recommended by the supplier.  Non-stranded RNA libraries were prepared by New England Biolabs NEB/NEXT SingleCell /Low Input Library Prep Kit exactly as recommended.