Single-cell RNA Sequencing of Fluorescently Labeled Mouse Neurons Using Manual Sorting and Double In Vitro Transcription with Absolute Counts Sequencing (DIVA-Seq)

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 droplets^1,2, 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 a 0.2 µm polyvinylidene difluoride (PVDF) membrane syringe filter and a two-way tubing valve using suitable tubing connectors.
3. Prepare

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...

Materials

Name	Company	Catalog Number	Comments
ERCC RNA Spike-In Control Mixes	Thermo Fisher	Cat# 4456740
SuperScript III	Thermo Fisher	Cat# 18080093
RNaseOUT Recombinant Ribonuclease Inhibitor	Thermo Fisher	Cat# 10777019
RNA fragmentation buffer	New England Biolabs	Cat# E6105S
RNA MinElute kit	Qiagen	Cat# 74204
Antarctic phosphatase	New England Biolabs	Cat# M0289
Poly nucleotide kinase	New England Biolabs	Cat# M0201
T4 RNA ligase2, truncated	New England Biolabs	Cat# M0242
Ampure Xp magnetic  beads	Beckman Coulter	Cat# A63880
SPRIselect size selection magnetic beads	Thermo Fisher	Cat# B23317
DL-AP5	Tocris	Cat# 0105
CNQX	Tocris	Cat# 1045
TTX	Tocris	Cat# 1078
Protease from Streptomyces griseus	Sigma-Aldrich	Cat# P5147
Message Amp II kit	Thermo Fisher	Cat# AM1751
Carbogen	Airgas	Cat# UN3156
Sylgard 184	Sigma-Aldrich	Cat# 761036
Illumina TrueSeq smallRNA kit	Illumina	Cat# RS-200-0012
Bioanalyzer RNA Pico chip	Agilent	Cat# 5067-1513
Bioanalyzer High Sensitvity DNA chip	Agilent	Cat# 5067-4626
Bioanalyzer 2100	Agilent
Dissection microscope with fluorescence and bright field illumination with DIC optics.  (Leica model MZ-16F).	Leica	Model MZ-16F
Glass microcapillary: Borosilicate capillary tubes 500/pk. OD= 1 mm, ID=0.58 mm, wall= 0.21 mm, Length= 150 mm.	Warner instruments	Model GC100-15, Order# 30-0017
Capillary pipette puller	Sutter Instruments Co	P-97
Vibratome	Thermo Microm	HM 650V
Vibratome tissue cooling unit	Thermo Microm	CU 65