Illumina NovaSeq 6000 paired end sequencing; Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry
Lineage plasticity in SCLC generates non-neuroendocrine cells primed for vasculogenic mimicry
Circulating tumour cells were collected from patient blood using RosetteSep (Stemcell Technologies) and injected into the flank of non-obese diabetic severe combined immunodeficient interleukin-2 receptor γ–deficient mice. Tumours were excised from terminally sacrificed mice, dissociated into single cells using Miltenyi Biotec's tumour dissociation kit (#130-095-929) following the manufacturer's instructions on a gentleMACS octo dissociator (Miltenyi Biotec, #130-096-427) and cultured ex vivo. Suspension and adherent cells were separated, cultured on either plastic or Matrigel for 24 hours and total RNA was harvested from cells using a RNeasy Mini Kit (Qiagen, #74106) according to the manufacturer's instructions. Total RNA was harvested from cells using a RNeasy Mini Kit (Qiagen, #74106) according to the manufacturer's instructions using ice cold RLT buffer with β-mercaptoethanol (Sigma-Aldrich, #M6250). On-plate lysis was performed for adherent cells whereas suspension cells were lysed as a cell pellet. For cell numbers >1 x 106, the lysed cells were homogenised using a QIAshredder column (Qiagen, #79654) and for cell numbers <1 x 106 cells were homogenised by vortexing. RNA was measured using a NanoDrop (Thermo Fisher Scientific) and stored at -80°C. Indexed PolyA libraries were prepared using 200 ng of total RNA with the Agilent SureSelect Strand Specific RNA Library Prep Kit for Illumina Sequencing (Agilent, #G9691B).