Oct 05, 2022
Single-cell RNA-seq for mDA neurons
- 1UCL Institute of Neurology
Protocol Citation: gurvir.virdi 2022. Single-cell RNA-seq for mDA neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr4dxpgmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 05, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70858
Keywords: ASAPCRN
Abstract
Harvesting and performing single-cell RNA-seq on hiPSC-derived mDA neurons.
Harvesting cells for RNA-seq
Harvesting cells for RNA-seq
5m
5m
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 00:05:00 at 37 °C .
5m
mDA neurons are collected as a single cell suspension and diluted 1:3.
Quality and concentration of cells
Quality and concentration of cells
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.
single-cell RNA-seq
single-cell RNA-seq
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.