Functional and Molecular Single-Cell Analyses Implicate PRDM14 in the Initiation of a Novel B-1 Cell Malignancy - Single Cell RNA sequencing experiments
Functional and Molecular Single-Cell Analyses Implicate PRDM14 in the Initiation of a Novel B-1 Cell Malignancy - Single Cell RNA sequencing experiments
Single-cell suspensions were prepared from pre-leukemic mouse bone marrow under sterile conditions. Red blood cells were lysed with Red Blood Cell Lysis Solution (Miltenyi Biotec) and washed with staining media [SM: Hanks' Balanced Salt Solution (Thermo Fisher Scientific), 2% fetal bovine serum (FBS, Wisent), 10mM HEPES, pH 7.2]. Depletion of CD11b+ cells was carried out by magnetic cell separation with autoMACS (Miltenyi Biotec) per manufacturer's instructions. Bone marrow cells were stained with fluorochrome-conjugated antibodies for 30 minutes in the dark at room temperature and CD8β- CD19+ CD127+ Sca-1+ were sorted on a MoFlo Astrios (Beckman Coulter) in the Flow Cytometry Facility at the Hospital for Sick Children. Polyinosinic:polycytidylic acid (pIpC) administration to activate Mx1-cre, and consequently Prdm14 expression, was performed at 8 weeks of age with one intraperitoneal injection of 250µg. The 10X Genomics Chromium platform was used for single cell RNA-sequencing (scRNA-seq) at the Princess Margaret Genomics Centre. Determination of the viability and density of sorted cells were carried out by a Countess Automated Cell Counter (ThermoFisher Scientific). Capture and encapsulation of cells (approximately 2,000 per sample) was followed by cDNA amplification and library preparation for sequencing.