Illumina NovaSeq 6000 paired end sequencing; RNA-seq of human breast cancer cell lines MCF7 and T74D naive and resistant to palbociclib treated with capivasertib, fulvestrant and combination
RNA-seq of human breast cancer cell lines MCF7 and T74D naive and resistant to palbociclib treated with capivasertib, fulvestrant and combination
A T25 flask was set up for each treatment with 7x105 cells/flask, n=3 independent samples for T47D, T47D RB-, T47D CDK6H and MCF7 PacqR. 5 samples were generated for MCF7 parental, MCF7 RB- as well as MCF7 PacqR continuous palbociclib only (to account for sequencing failures). Cells were incubated at 37°C, 5% CO2 for 96 hours in the absence of palbociclib or with continuous palbociclib treatment in PalboR cells as indicated. RNA extraction was carried out using the RNeasy kit protocol (Qiagen, Manchester) and the resultant samples sent for RNA-sequencing at Novogene (Cambridge, UK) for the T47D cell lines and Genewiz/Azenta for the MCF7 parental and MCF7 RB- as well as MCF7 PacqR continuous palbociclib only. All MCF7 PacqR samples were sequenced internally at AstraZeneca. Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England BioLabs; #E7760L) or NEBNext® Ultra™ II RNA Library Prep Kit (New England BioLabs; E7770L) as per manufactuer's guidelines.