GSM9017408: MDAMB361 cell line, palbociclib resistant; Homo sapiens; RNA-Seq
Cells were encapsulated into nanoliter droplets and single cell transcriptomes barcoded using Bio-Rad ddSEQ Single Cell Isolator 2 (Bio-Rad). Briefly, to obtain a single cell suspension, a 60-90% confluent 25 cm2 plate of MDA-MB-361 was trypsinized, filtered through a 35µm nylon strainer (Falcon) and diluted to 2500 cells/µl in 1XPBS (Gibco) + 0.1 BSA (Santa Cruz Biotechnology) solution. We only proceeded with samples showing > 95% viability, according to the manufacturer's recommendations. Cell count and viability were evaluated with phase contrast microscope (Leica) and automated cell counter (Invitrogen). Finally, 4.5 µl of the cell suspension were loaded in each well of one ddSEQ M cartridge (Bio-Rad) in order to obtain an average of 300 single cells per chamber. cDNA library preparation was performed following the Illumina Bio-Rad SureCell WTA 3' Library kit (Illumina). Quantification and quality analysis of cDNA libraries were carried out on Agilent 2100 Bioanalyzer system (Agilent) using High Sensitivity DNA chips (Agilent). Libraries were sequenced on a Hiseq2500 Rapid Run Mode at Centre for Integrative Biology (CIBIO) Next Generation Sequencing (NGS) Facility, Trento, Italy, following protocol recommendations (Illumina); The bases per reads were: R1 (68) - I (8) - R2 (75); 10% PhiX per lane was included and R1 Custom Seq Primer was spiked in HP10.