Single-Cell Library Preparation of iPSC-Derived Neural Stem Cells

Abstract

Single-cell RNA-seq technology allows for the identification of heterogeneous cell populations, measures stochastic gene expressions, and identifies highly variable genes. Thus, with this technology it is possible to identify relevant pathways involved in development or in disease progression. Herein, we describe a protocol to capture and process single-cell transcriptomes that will be used for RNA sequencing. This chapter discusses the use of the Fluidigm C1 System and Integrated Fluidic Circuit microfluidics system, TapeStation 4200, SMART-Seq v4, Nextera XT Library Preparation Kit, and AMPure XP beads.

Keywords: C1; Fluidigm; Library preparation; Neural stem cells; Next-generation sequencing; Nextera XT; RNA-seq; SMART-Seq; Single cell; Single-cell RNA-seq.

Fig. 1

Workflow for single-cell RNA-seq using iPSC-derived NSCs with the C1 auto prep system. NSCs derived from iPSCs grow as neurospheres. Neurospheres are dissociated into single cells and loaded into the C1 IFC. Following the single-cell cDNA generation, libraries are amplified and indexed with the Nextera XT Library Preparation Kit. Generated libraries are then cleaned with AMPure XP beads and quality checked with the Agilent TapeStation 4200. Cleaned libraries are then sequenced with an Illumina-based next-generation sequencing platform. Raw reads are then demultiplexed and analyzed

Fig. 2

Integrated Fluidic Circuits (IFC) priming reagent map

Fig. 3

IFC loading cell map

Fig. 4

IFC sample prep map

Fig. 5

Harvesting sequence map