GSM9311054: AMO-1/CFZ cell, P-gp HIGH, rep3; Homo sapiens; RNA-Seq
For RNA sequencing, the carfilzomib-resistant AMO-1/CFZ cells were stained for P-glycoprotein expression for subsequent sorting. Cells were stained with a FITC-conjugated mouse monoclonal antibody against human P-glycoprotein (clone 17F9; BD Biosciences, USA) for 30 minutes in the dark without prior fixation and then washed twice with PBS. Control AMO-1 cells (P-glycoprotein non-expressing) were used for RNA sequencing as an unsorted total lysate. Analysis and sorting of AMO-1/CFZ cells were performed on a FACSAria SORP instrument (BD Biosciences, USA). Based on the P-glycoprotein fluorescence intensity, stained AMO-1/CFZ cells were sorted into two distinct subpopulations: P-gp LOW and P-gp HIGH. The purity of the isolated subpopulations was confirmed by immediate post-sort analysis. Four independent biological replicates were performed. Data were acquired using BD FACSDiva 8.1.1 software and analyzed using FlowJo software (BD Biosciences, USA). RNA libraries were synthesized using the NR605-02 VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (VAZYME, China) with indexed barcodes from the N809 VAHTS RNA Adapters Set 3 for Illumina (VAZYME, China) according to the manufacturer's protocols. Poly(A)+ RNA selection was performed with the N403-02 VAHTS mRNA Capture Beads 2.0 (VAZYME, China). RNA quality control was carried out using the C405110 Qsep400, R1 Cartridge (RNA Cartridge) (Bioptic Inc., Taiwan), and the quality of the final libraries was assessed with the C405105 Qsep400, N1 Cartridge (High Sensitivity Cartridge) (Bioptic Inc., Taiwan).