title: RNA-sequencing of treatment-naive, Lapatinib-resistant, and resistant-reversal SUM149 IBC cells
design: RNA-sequencing of treatment-naive, Lapatinib-resistant, and resistant-reversal SUM149 IBC cells
library_construction_protocol: Monolayer cells of SUM149, rSUM149, and rrSUM149 (n=3 for each cell line) seeded with their respective medium in 100 mm dishes (250,000 cells/dish) were harvested 72 hours after they were seeded. Monolayer cells were lifted using a cell scraper instead of trypsin-EDTA. After collection, monolayer cells were pelleted, washed with ice-cold PBS, and re-pelleted to be stored at -80°C until RNA extraction.  SUM149PT cells were obtained from Asterand Inc. and were routinely cultured in Ham's F12 medium supplemented with 5% FBS, 1% penicillin/streptomycin, 1% antibiotic/antimycotic, hydrocortisone and an insulin/transferrin/selenium cocktail. To generate the resistance model rSUM149, SUM149 cells were routinely maintained in normal culture medium supplemented with 7.5 μM research grade Lapatinib in increasing concentrations (0.25– 7.5 μM) for a period of 3 months. Cells were grown for two weeks in each dosage concentration. Following an initial decrease in cell growth and cell death, small colonies of viable cells were seen by the end of the two weeks and were cultured to confluence before the next drug concentration increase. The resistance reversal model rrSUM149 was generated from rSUM149 after removal of Lapatinib >3 months and were cultured in regular SUM149 media. All cell lines were cultured at 37C with 5% CO2 for no more than 2 months prior to use in this study. Total RNA was extracted was conducted using Qiagen RNeasy Plus Universal mini kit following the manufacturer's instructions. Extracted RNA samples were quantified using Qubit 2.0 Fluorometer and RNA integrity was checked using Agilent TapeStation 4200. RNA sequencing libraries (n=9) were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following the manufacturer's instructions. Briefly, mRNAs were enriched with Oligo(dT) beads and fragmented for 15 minutes at 94 °C. First strand and second strand cDNAs were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation, and quantified by using Qubit 2.0 Fluorometer as well as by quantitative PCR.