Carbonic anhydrase IX (CA9)-positive tumor cells were     immunomagnetically purified (using Miltenyi anti-CA9 PE antibody and anti-PE     microbeads) from freshly dissociated tumor, and cultured for 48-72 hours in OptiMEM     GlutaMax media (Gibco) containing 5% fetal bovine serum, 1mM sodium pyruvate, 100U     mL penicillin and streptomycin, 50µg mL gentamicin, 5µg mL insulin, and 5ng mL EGF.     RNA was extracted using the Qiagen RNeasy kit, and RNA-sequencing of this low-input     RNA was performed using a modified version of the Smart-seq2 protocol (using NEBNext     Single Cell Low Input cDNA Synthesis & Amplification module), as previously     described.