CAMA1 cells RNAseq

CAMA1 cells were plated in full media ± 2 ng/mL FGF2 (Sigma) and then treated with DMSO or 1 mM fulvestrant plus 1 mM palbociclib ± 1 mM lucitanib for 6 h. Cells were harvested and RNA was purified using a RNA purification kit (Maxwell, Promega). Total RNA was quantified using the Quant-iT Ribo-Green RNA Assay Kit (Invitrogen) and normalized to 4 ng/mL; 200 ng of each sample were used for library preparation in an automated variant of the Illumina Tru Seq RNA Sample Preparation protocol (Revision A, 2010). This method uses oligo(dT) beads to select mRNA from the total RNA sample and is followed by heat fragmentation and cDNA synthesis from the RNA template. The resultant cDNA went through library preparation (end repair, base "A" addition, adapter ligation, and enrichment) using Broad Institute–designed indexed adapters for multiplexing. After enrichment, libraries were quantitated with qPCR using the KAPA LibraryQuantification Kit for Illumina Sequencing Platforms and pooled equimolarly. The entire process was performed in a 96-well format with all pipetting done by either the Agilent Bravo or PerkinElmer JANUS Mini liquid handlers.<br>Sample descriptions are as below:<br>170-LF-1_S1 CAMA1 CNT1 PLUS FGF STIMULATION 170-LF-2_S2 CAMA1 CNT2 PLUS FGF STIMULATION 170-LF-3_S3 CAMA1 CNT3 PLUS FGF STIMULATION 170-LF-10_S10 CAMA1 FULVESTRANT+PALBOCICLIB 1 PLUS FGF STIMULATION 170-LF-11_S11 CAMA1 FULVESTRANT+PALBOCICLIB 2 PLUS FGF STIMULATION 170-LF-12_S12 CAMA1 FULVESTRANT+PALBOCICLIB 3 PLUS FGF STIMULATION 170-LF-13_S13 CAMA1 FULVESTRANT+PALBOCICLIB+LUCITANIB 1 PLUS FGF STIMULATION 170-LF-14_S14 CAMA1 FULVESTRANT+PALBOCICLIB+LUCITANIB 2 PLUS FGF STIMULATION 170-LF-15_S15 CAMA1 FULVESTRANT+PALBOCICLIB+LUCITANIB 3 PLUS FGF STIMULATION 170-LF-16_S16 CAMA1 CNT1 170-LF-17_S17 CAMA1 CNT2 170-LF-18_S18 CAMA1 CNT3<br>