GSM9271824: MGG123_Mouse3_BloodVessel PDX; Homo sapiens; RNA-Seq
Tissue from each location were mechanically and enzymatically dissociated using a papain-based neural tissue dissociation kit (Miltenyi Biotec) supplemented with 0.1% Type I collagenase (Thermo Fisher Scientific)/PBS. The dissociated cells were first stained with calcein Blue AM (BD Biosciences) and Zombie NIR (BioLegend) for 25 min at 4C, and with anti-mouse CD16/32 (BD Biosciences) for 5 min. After washing cells with ice-cold 1% BSA/PBS, the cells were then stained with PerCP anti-mouse CD45 (clone 30-F11, BD Biosciences) for 30 min at 4C. Sorting was performed with Becton Dickinson Influx cytometer (Becton Dickinson). Side scatter (SSC) width versus forward scatter (FSC) area and Trigger Pulse Width versus FSC criteria were used to discriminate doublets and gate-only singleton cells. Viable single cells were identified as calcein blue AM positive and Zombine NIR negative to low cells. We sorted viable CD45 negative/GFP positive cells from blood-vessel associated invasion samples into 96-well plates containing 10uL of TCL buffer (QIAGEN) with 1% beta-mercaptoethanol (Sigma Aldrich). Plates were frozen immediately after sorting and stored at -80 degree prior to whole transcriptome amplification, library preparation, and sequencing. Smart Seq 2