GSM5746454: KP tumour cell line 2_FOSL1_ChIP_rep1; Mus musculus; ChIP-Seq
Mouse KP cell lines were cultured in DMEM (Thermo Fisher) with 10% FBS and 1% Antibiotic-Antimycotic. Cultured cells were trypsinised and filtered through a 70 µm filter to obtain a single cell suspension. To obtain primary SK tumour cells, 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 inflated with lung/tumour dissociation buffer (Miltenyi Biotec), cut into small pieces and incubated at 370C for 45 minutes. 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). tdTomato+ tumour cells were sorted using Sony SH800 sorter. Approximately 500,000 cells were used from each sample. FOSL1 binding sites were identified using the Mint-ChIP assay (Kobayashi et al., 2020; van Galen et al., 2016). Updated version of the Mint-ChIP protocol (version 3) was used (https://tinyurl.com/udqksct). Cells were lysed on ice for one hour followed by digestion with 300 units of MNase for 10 minutes at 370C. T7 adapters were then ligated, and samples were incubated with anti-FOSL1 antibody overnight at 40C. Chromatin fragments bound to antibody were isolated using protein A/G Dynabeads and digested with proteinase K for one hour at 630C. DNA was purified using 1x SPRI bead clean-up. In vitro transcription was performed using T7 RNA polymerase (New England BioLabs). RNA was purified using silane beads followed by reverse transcription using Superscript III enzyme (Thermo Fisher). cDNA was purified using SPRI beads and used for library preparation PCR using Terra DNA polymerase (Takara). Size selection of libraries was performed using SPRI beads. Libraries were sequenced using NovaSeq S4 flow cell in 150 bp PE configuration. Mint-ChIP