Genomics chromium v2 kit

Human first trimester subcortical forebrain tissue was obtained from elective routine abortions (10 weeks postconception) with the written informed consent of the pregnant woman and in accordance with the ethical permit given by the Regional Ethics Vetting Board (Stockholm, Sweden). Human fetal forebrain tissue was collected and stored in hibernation media with addition of GlutaMAX and B-27 supplements according to the manufacture’s instructions (overnight, 4oC, Hibernate-A, Thermo-Fisher). The tissue was then cut into small cubic pieces of approximately 1-2mm length. Tissue was dissociated using a dissociation kit (Miltenyi, Neural Tissue Dissociation Kit (P)) according to manufacture’s instructions. In short, tissue was prepared in the kit buffer containing 0.067mM beta-mercaptoethanol. After addition of enzyme mix 1 and 2, the tissue was mechanically dissociated using three increasingly smaller gauges of fire polished Pasteur pipettes, pipetted 20, 15 and 10 times up and down respectively. Ultimately, collected cells were stored on ice in PBS containing 1% BSA and immediately prepared for single cell library preparation. Single-cell RNA sequencing was performed using the 10X Genomics Chromium V2 kit, following the manufacturer’s protocol, and sequenced on an Illumina Hiseq 2500.