Illumina HiSeq 4000 paired end sequencing; Single-cell RNA-seq of the human fetal lateral ganglionic eminence at 7, 9 and 11 post-conceptional weeks
Single-cell RNA-seq of the human fetal lateral ganglionic eminence at 7, 9 and 11 post-conceptional weeks
Embryonic and fetal age was extrapolated based on the date of the mother's last menstruation, ultrasound scans of the fetus in utero, crown-rump length (CRL) and visual inspection. Depending on the condition and period of the procured specimens the LGE was dissected. Both left and right brain regions of interest were collected from fresh tissues. Specimens were chilled on ice during dissection. Dissected LGEs were maintained in Hibernate-E media (Thermo Fisher, A1247601).   Tissues were processed by dissociation using Papain Dissociation System (Worthington, LK003150) following the manufacturer's recommendations, adjusting the incubation time based on tissue piece size. Briefly, after papain incubation, cells were resuspended in N2 media with DNaseI provided in Papain Dissociation System (Worthington, LK003170) and Rock Inhibitor Y-27632 (provided by CHDI Foundation, CHDI-00197406-0001-007). Next, glass pipettes of increasingly smaller tip diameter (fire-polished) were used to dissociate the tissue to a single-cell suspension followed by a filtration with CellTrics 20um (Sysmex, 04-004-2325). Cells were pelleted and resuspended in Ultrapure BSA 0.04% (Ambion, AM2616). The Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3′ Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3′ Reagents Kits V2 User Guide.  The Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN- 120237), Chromium Single Cell 3′ Chip kit v2 (PN-120236) and Chromium i7 Multiplex Kit (PN-120262) were used according to the manufacturer's instructions in the Chromium Single Cell 3′ Reagents Kits V2 User Guide.