Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.

Abstract

Single-cell RNA sequencing (RNA-seq) is now a widely implemented tool for assaying gene expression. Commercially available single-cell RNA-sequencing platforms process all input cells indiscriminately. Sometimes, fluorescence-activated cell sorting (FACS) is used upstream to isolate a specifically labeled population of interest. A limitation of FACS is the need for high numbers of input cells with significantly labeled fractions, which is impractical for collecting and profiling rare or sparsely labeled neuron populations from the mouse brain. Here, we describe a method for manually collecting sparse fluorescently labeled single neurons from freshly dissociated mouse brain tissue. This process allows for capturing single-labeled neurons with high purity and subsequent integration with an in vitro transcription-based amplification protocol that preserves endogenous transcript ratios. We describe a double linear amplification method that uses unique molecule identifiers (UMIs) to generate individual mRNA counts. Two rounds of amplification results in a high degree of gene detection per single cell.

Introduction

Single-cell RNA sequencing (RNA-seq) has transformed transcriptomic studies. While large-scale single-cell RNAseq can be performed using a variety of techniques, such as droplets1,2, microfluidics3, nanogrids4, and microwells5, most methods cannot sort defined cell types that express genetically encoded fluorophores. To isolate a select cell population, fluorescence-activated cell sorting (FACS) is often used to sort labeled cells in a single-cell mode. However, FACS has some restrictions and requires meticulous sample processing steps. Fi....

Protocol

All the procedures including animal subjects have been approved by IACUC at Cold Spring Harbor Laboratory, NY (IACUC #16-13-09-8).

1. Manual Sorting of Fluorescently Labeled Mouse Neurons

  1. Pull glass microcapillaries (see Table of Materials) to 10–15 µm exit diameter using a capillary puller with the following settings: heat: 508, pull: blank, vel: blank, time: blank.
  2. Attach 120–150 cm flexible silicone tubing (~0.8 mm inside diameter) to .......

Representative Results

Using the protocol described above, GABAergic neurons were manually sorted (Figure 1) and RNA was amplified, then made into a cDNA library (Figure 2) and sequenced at high depth8. The amplified RNA (aRNA) products ranged between 200–4,000 bp in size, with a peak distribution slightly above 500 bp (Figure 3A). The bead-purified cDNA library was further size-restricted by .......

Discussion

The manual sorting protocol is suitable for a supervised RNA sequencing of neuron populations that are either sparsely labeled in the mice brain or are representing a rare cell population that is otherwise not feasible to study using current high-throughput cell sorting and amplification methods. Cells subjected to FACS usually undergo sheath and sample line pressures in the range of ~9–14 psi, depending on nozzle size and desired event rates. In addition, upon being ejected from the nozzle, the cells can land hard.......

Acknowledgements

This work was supported by grants from the NIH (5R01MH094705-04 and R01MH109665-01 to Z.J.H.), by the CSHL Robertson Neuroscience Fund (to Z.J.H.), and by a NARSAD Post-Doctoral Fellowship (to A.P.).

....

Materials

NameCompanyCatalog NumberComments
ERCC RNA Spike-In Control MixesThermo FisherCat# 4456740
SuperScript IIIThermo FisherCat# 18080093
RNaseOUT Recombinant Ribonuclease InhibitorThermo FisherCat# 10777019
RNA fragmentation bufferNew England BiolabsCat# E6105S
RNA MinElute kitQiagenCat# 74204
Antarctic phosphataseNew England BiolabsCat# M0289
Poly nucleotide kinaseNew England BiolabsCat# M0201
T4 RNA ligase2, truncatedNew England BiolabsCat# M0242
Ampure Xp magnetic  beadsBeckman CoulterCat# A63880
SPRIselect size selection magnetic beadsThermo FisherCat# B23317
DL-AP5TocrisCat# 0105
CNQXTocrisCat# 1045
TTXTocrisCat# 1078
Protease from Streptomyces griseusSigma-AldrichCat# P5147
Message Amp II kitThermo FisherCat# AM1751
CarbogenAirgasCat# UN3156
Sylgard 184Sigma-AldrichCat# 761036
Illumina TrueSeq smallRNA kitIlluminaCat# RS-200-0012
Bioanalyzer RNA Pico chipAgilentCat# 5067-1513
Bioanalyzer High Sensitvity DNA chipAgilentCat# 5067-4626
Bioanalyzer 2100Agilent
Dissection microscope with fluorescence and bright field illumination with DIC optics.  (Leica model MZ-16F).LeicaModel MZ-16F
Glass microcapillary: Borosilicate capillary tubes 500/pk. OD= 1 mm, ID=0.58 mm, wall= 0.21 mm, Length= 150 mm.Warner instrumentsModel GC100-15, Order# 30-0017
Capillary pipette pullerSutter Instruments CoP-97
VibratomeThermo MicromHM 650V
Vibratome tissue cooling unitThermo MicromCU 65

References

  1. Macosko, E. Z., et al. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell. 161 (5), 1202-1214 (2015).
  2. Klein, A. M., et al. Dropl....

Explore More Articles

Single cell RNA SequencingFluorescently Labeled NeuronsManual SortingDouble In Vitro TranscriptionDIVA SeqNeuron IsolationSubpopulationFACS SortingGlass MicrocapillariesArtificial Cerebrospinal FluidProteaseFBSPasteur PipettesBrain DissectionVibratomeLabeled CellsActivity Blockers

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved