Illumina HiSeq 4000 paired end sequencing; Spatiotemporal dynamics of CRC metastases resolved by single cell RNAseq
Primary tumours and micro-dissected liver metastases were chopped with razor blades. Subsequent enzymatic digestion was performed with 200 U/ml collagenase IV (Sigma Aldrich) in HBSS (Lenovo) for 30 min at 37 °C, in a shaking water bath or a gentleMACS Dissociator (Miltenyi Biotec). Digested tissue fragments were then filtered through 100- and 40-μm meshes, washed and treated for 5 minutes with ammonium chloride. Single-cell preparations were first blocked with anti-CD16/32 (clone 93; eBioscience) and then stained with APC anti-Epcam (118214; Biolegend) and BV605 anti-CD45 (103155, Biolegend) antibodies. Finally, cells were resuspended in HBSS with 0.5% FBS and DAPI (Sigma Aldrich). For isolation of low numbers of tumour cells from mice without visible metastases, whole livers were thoroughly minced with razor blades. After an initial 30-minute digestion with 200 U/ml collagenase IV, samples were filtered through 100 μm meshes. Although most cells flowed through, a small fraction of the samples –highly enriched for tumour cells- was retained in the filter (Supplementary Figure 5c). Filters were then laid into a 6-well plate and covered with HBSS containing collagenase IV, Dispase II and DNase I. After re-digestion in a water bath for 30 minutes al 37 °C, most cells now seeped through the filters. The protocol continued with washing, ammonium chloride treatment. Freshly harvested single cells were sorted into 96-well plates containing 2.5 µl of the Reaction buffer (1x Lysis Buffer, RNase Inhibitor 1U/µl).  Full-length single-cell RNA-seq libraries were prepared using the SMART-Seq v5 Ultra Low Input RNA Kit for Sequencing (Takara Bio). All reactions were downscaled to one quarter of the original protocol and performed following thermal cycling manufacturer's conditions.