Instrument: AB 5500xl Genetic Analyzer
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: To obtain single cell suspension, PDXs were mechanically and then enzymatically digested in a solution of collagenase IV (5mg/ml) and DNase (100U/ml) (Sigma-Aldrich) in DMEM/F12 (Lonza) for 1h at 37 °C. Partially digested tissue was filtered through a 100µm cell strainer (BD Falcon) and red blood cells were removed by Lysing Buffer 1X (BD Bioscience). Single cell suspensions from dissociated NSCLC-PDXs were washed and incubated in staining solution 1% BSA and 2mM EDTA with specific antibodies at appropriate dilutions for 30 min at 4 °C: PE anti-human CD133/1, FITC anti-human CD326 (EpCAM) (Miltenyi Biotech), APC anti-human CD187 (CXCR4) (BD Pharmingen), Alexa Fluor® 488 anti-human HLA-ABC (BD Pharmingen) , PerCP-eFluor 710 anti-mouse MHC class I (e-Bioscience). Prior to sorting, cells were resuspended to a final concentration of 10x106 cells/ml in Hepes Buffered Saline (HBS) (Lonza) + 0.1% B27 Supplements (Gibco) and 7-AAD viability staining solution (1:10) (e-Bioscience) for dead cells exclusion. PDX-cells were sorted with FACSAria (Becton Dickinson) into a chilled 96-well plate (Corning Incorporated). For sorting of different CSC fractions, an initial gate excluding doublets, dead cells and mouse MHC class I+ cells was set. Then, within the gate of human viable CD133+ cells, the fraction of EpCAM+ and CXCR4+EpCAM- cells were identified and sorted. Total tumor population was isolated based on human HLA-ABC+ expression. For each cell fraction, 300 cells were directly sorted in 30 μl of CLS (Complete Lysis Solution) per well and within 1h approximately 100 cells/10 μl were transferred into 0.5 ml tubes and stored to -80 0C. To ensure all cDNA generated using the Epistem RNA Amp kit was double stranded one cycle of re-amplification was performed. RNA (1ng-25pg) or cell lysates in CLS were adjusted to 6.75ml volume and RNA-amplified using the EpiStem RNA-Amp Kit according to the manufacturers protocols (EpiStem, Manchester, UK). Briefly the samples underwent oligo-dT priming and 5’ capping prior to x35 cycles of PCR amplification using the conditions 90ºC 30 sec, 42ºC 2 min and 72ºC 6 min. Following amplification all samples were purified using a MoBio UltraClean® PCR Clean-Up Kit (Carlsbad, CA, USA) and quantified on a NanoDrop spectrophotometer.