GSM1972123: single cell RNA-seq [NPC3_S23]; Mus musculus; RNA-Seq
Timed-pregnant mice were sacrificed by deep anesthesia followed by cervical dislocation. The embryos were quickly removed and cortical hemispheres were isolated, ganglionic eminences removed, and all pups brains were pooled. Single cells were isolated by gentle trituration in ice-cold phosphate buffered saline containing 2 mM EGTA (PBSE) using P1000 tips with decreasing bore diameter. Cells were then filtered through a 40 uM nylon cell strainer and stained with propidium iodide (PI), a live-dead stain, and fluorescence activated single cell sorting (FACS) was performed selecting for PI negative cells. Samples remained on ice throughout the process and total processing time from cervical dislocation to sorting was limited to 2 hours. Single cells were sorted directly into cell lysis buffer provided in the Clontech SMARTer® Ultra™ Low RNA Kit for Illumina® Sequencing (cat # 634936), and sequencing libraries were generated using the manufacturer’s protocol. Resulting libraries were sequenced on the Illumina® HiSeqTM 2000 sequencing platform.