GSM6543825: mouse breast cancer multiome (scRNA-seq + scATAC-seq) late stage biological replicate 1; Mus musculus; OTHER
Tumor tissue specimens from mouse or human specimens was minced on ice. The tissue was then digested for 3x 30 minutes in a 37C water bath agitator in 0.5 mg/mL collagenase (collagenase type IV, ThermoFisher) digest buffer in Medium 199 (HyClone, GE Healthcare) consisting of 5% fetal bovine serum (Gibco FBS, ThermoFisher), DNase I (Worthington), Poloxamer 188 (Cat. P5556-100ML, Sigma), HEPES, and CaCl2. The digest was quenched with quench media (DMEM (Gibco DMEM, ThermoFisher) with 15% FBS), then centrifuged at 300 x G for 5 minutes at 4C, resuspended in quench media, and filtered through 100, 70, and 40m cell strainers (Falcon cell strainer, ThermoFisher). Red blood cell lysis was performed using Hybri-Max (Sigma) per the manufacturer's protocol. Cells were counted and re-suspended for further processing. Multiome was then performed at the Stanford Genomics Facility (SGF) using the 10x Genomics platform. The relevant protocols were followed per manufacturer's guidelines. Nuclei isolation was pursued as for scATAC-seq sequencing using 0.1x lysis buffer with 3-minute incubation. Final libraries were analyzed on an Agilent Bioanalyzer High Sensitivity DNA chip for qualitative control purposes. Libraries were sequenced on a HiSeq 4000 Illumina platform targeting 50,000 reads per cell.