GSM5573031: PDX_Epcam+_14-405; Homo sapiens; ATAC-seq
Mice were anaesthetised using Avertin. Skin and muscular layers were cut to expose the abdominal and thoracic cavities. Descending aorta was cut to exsanguinate and euthanise the animal. Cold PBS was perfused through the cardiac right ventricle to flush the blood out of the lungs. Lungs were then removed and inflated with 4% PFA and fixed overnight at 40C in PFA for histology. For isolation of live primary cells, lungs were instead inflated with lung/tumour dissociation buffer (Miltenyi Biotec), cut into small pieces and incubated at 370C for 30 minutes (normal lung) or 45 minutes (lung with neoplastic growth). A single cell suspension was obtained by mechanically dissociating using gentleMACS (Miltenyi Biotec) dissociator and passing the samples through a 70 µm filter. RBCs were lysed by resuspending the cells in ACK lysis buffer and leucocytes were depleted by using anti-CD45 microbeads (Miltenyi Biotec). To isolate AEC2 cells from CC mice, Lysotracker dye was used as described in (der Velden et al., 2013). Briefly, dissociated lung cells were resuspended in DMEM/F12 containing Lysotracker (1 µL dye in 14 mL medium) and incubated at 370C for 30 minutes. Human normal lung and cancer samples were stored on ice in DMEM/F12 medium supplemented with 2% FBS until dissociation. Samples were dissociated using human tumour dissociation kit (Miltenyi Biotec) following the manufacturer's recommendation. A single cell suspension was obtained by passing the dissociated samples through a 70 µm filter. RBCs were lysed by resuspending the cells in ACK lysis buffer and leucocytes were depleted by using anti-CD45 microbeads (Miltenyi Biotec). Samples were resuspended in PBS supplemented with 10% FBS and incubated with anti-EpCAM antibodies for 10 minutes at room temperature. Samples were then washed once in PBS supplemented with 10% FBS. Primary cells were isolated by FACS using SONY SH800 sorter. Sytox dyes (Blue, Green, Red and AADvanced) were used to exclude dead cells. Harvested primary cells were immediately used to prepare ATAC-seq libraries as described in (Buenrostro et al., 2013). Briefly, isolated cells were washed in PBS and resuspended in buffer containing transposase Tn5 and incubated at 370C for 30 minutes. Tagmented DNA was PCR amplified for 10-16 cycles. Mitochondrial DNA present in the libraries was cleaved using a CRISPR-Cas9 based method described in (Wu et al., 2016). sgRNA library targeting mitochondrial DNA was purchased from Addgene (#82480). In vitro transcription was performed to produce the gRNAs using MEGAshortscript Kit (Thermo Fisher Scientific). ATAC-seq libraries were treated with 10 µg Cas9 protein and 15 µg of sgRNA in a 40 µL reaction for 2 hours at 370C. The libraries were purified using Qiaquick PCR purification kit (Qiagen) and sequenced using Illumina Hi-Seq 2500 (50 bp paired end) or Next-Seq (75 bp paired end).