title: GSM6919185: MEKi_rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Slow-frozen PDX tissue samples were quickly thawed in the water bath set to 37°C degree, re-suspended in 10 mL of ice cold RPMI with 1% BSA  and spun down at 300g for 5 min. Further tissue samples were cut into small pieces and enzymatically digested for 30 min at 37 °C on a shaker with 5000U Collagenase IV, 15 KU DNAse I, 2 mL Accutase dissolved in RPMI with 2mM CaCl2 and 0.5% BSA 5 mL RPMI mixture. After incubation, digested tissue was filtered through 100 μm nylon and then through 35-μm cell strainers. For samples with viability below 80%, apoptotic and dead cells were removed using immunomagnetic cell separation with the Annexin Dead Cell Removal Kit and EasySepTM Magnet. If the cell pellet appeared red, red blood cell lysis was performed following the manufacturer's instructions. Cell number and viability was assessed on Luna-FLTMDual Fluorescence Cell counter using Photon slides by acridine orange propidium iodide stain and optimal cell concentrations were set to 700-1100 cells/μL according to 10x Genomics protocols. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). 6000 cells per sample were targeted and libraries were diluted to 10 nM and pooled at balanced ratios according to the target cell number. Paired-end sequencing was performed on the Illumina NovaSeq S2 flow cell. According to 10x Genomics recommendation 50,000 read pairs per cell for GEX coverage was targeted.