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>

将CAMA1细胞接种于含或不含2 ng/mL成纤维细胞生长因子2（FGF2，Sigma）的完全培养基中，随后分别用二甲基亚砜（DMSO）、1 mM氟维司群联合1 mM帕博西尼，或在此基础上±1 mM卢卡替尼处理6小时。收集细胞后，使用RNA纯化试剂盒（Maxwell，Promega）纯化总RNA；采用Quant-iT Ribo-Green RNA检测试剂盒（Invitrogen）对总RNA进行定量，并将浓度校准至4 ng/mL。每份样本取200 ng用于文库构建，流程采用Illumina TruSeq RNA样本制备方案（修订版A，2010）的自动化变体版本。该方法通过寡聚(dT)磁珠从总RNA中富集mRNA，随后进行热片段化，并以RNA为模板合成cDNA。所得cDNA使用布罗德研究所（Broad Institute）设计的带索引接头进行多重测序，依次完成末端修复、碱基"A"加尾、接头连接及富集等文库制备步骤。富集完成后，采用适配Illumina测序平台的KAPA文库定量试剂盒通过qPCR对文库进行定量，并按等摩尔浓度混合所有文库。整个实验流程均采用96孔板体系，所有移液操作均由安捷伦（Agilent）Bravo或珀金埃尔默（PerkinElmer）JANUS Mini液体处理工作站完成。<br>样本描述如下：<br>170-LF-1_S1：CAMA1细胞对照组1，经FGF刺激<br>170-LF-2_S2：CAMA1细胞对照组2，经FGF刺激<br>170-LF-3_S3：CAMA1细胞对照组3，经FGF刺激<br>170-LF-10_S10：CAMA1细胞经氟维司群+帕博西尼处理1，且经FGF刺激<br>170-LF-11_S11：CAMA1细胞经氟维司群+帕博西尼处理2，且经FGF刺激<br>170-LF-12_S12：CAMA1细胞经氟维司群+帕博西尼处理3，且经FGF刺激<br>170-LF-13_S13：CAMA1细胞经氟维司群+帕博西尼+卢卡替尼处理1，且经FGF刺激<br>170-LF-14_S14：CAMA1细胞经氟维司群+帕博西尼+卢卡替尼处理2，且经FGF刺激<br>170-LF-15_S15：CAMA1细胞经氟维司群+帕博西尼+卢卡替尼处理3，且经FGF刺激<br>170-LF-16_S16：CAMA1细胞对照组1<br>170-LF-17_S17：CAMA1细胞对照组2<br>170-LF-18_S18：CAMA1细胞对照组3