Single-cell RNA-seq for mDA neurons

At the desired age of mDA neurons, they are harvested for single-cell RNA-seq:

mDA neurons are washed 1x in PBS.

They are incubated with Accutase (Thermo Fisher Scientific) for 0h 5m 0s at 37°C .

mDA neurons are collected as a single cell suspension and diluted 1:3.

The quality and concentration of each single-cell suspension was measured using Trypan blue and the Eve automatic cell counter.

Each sample presented a concentration between a 1200-1700 cell/µl and viability ranged between 55-68%, samples with a viability above 57% were used for sequencing.

Approximately 10000 cells were loaded for each sample into a separate channel of a Chromium Chip G for use in the 10X Chromium Controller (cat: PN-1000120).

The cells were partitioned into nanoliter scale Gel Beads in emulsions (GEMs) and lysed using the 10x Genomics Single Cell 3′ Chip V3.1 GEM, Library and Gel Bead Kit (cat: PN-1000121).

cDNA synthesis and library construction were performed as per the manufacturer’s instructions.

The RNA was reversed transcribed and amplified using 12 cycles of PCR.

Libraries were prepared from 10µl of the cDNA and 13 cycles of amplification. Each library was prepared using Single Index Kit T Set A (cat: PN-1000213) and sequenced on the HiSeq4000 system (Illumina) using 100 bp paired-end run at a depth of 65-100 million reads. Libraries were generated in independent runs for the different samples.