Feb 03, 2025

Public workspaceSingle-cell transcriptome library preparation

DOI
dx.doi.org/10.17504/protocols.io.e6nvwb532vmk/v1
  • 1Institut Imagine
  • Team Deleidi
Federico Bertoli
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DOI: dx.doi.org/10.17504/protocols.io.e6nvwb532vmk/v1
Protocol Citation: Maria Jose Perez J. 2025. Single-cell transcriptome library preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwb532vmk/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: November 26, 2024
Last Modified: February 03, 2025
Protocol Integer ID: 112838
Funders Acknowledgements:
ASAP
Abstract
Single-cell transcriptome library preparation
Single-cell transcriptome library preparation
Single-cell transcriptome library preparation
Dissociate iPSC-derived cells in the triculture system into single-cell suspensions by incubating with Accutase for 15 minutes at 37°C.
Filter the dissociated cell suspensions through a 40 μm filter.
Resuspend the filtered cells in PBS containing 0.04% BSA at a final concentration of 1000 cells/μL. Ensure that cell viability exceeds 95%.
Generate single-cell RNA-Seq (scRNA-Seq) libraries using the 10X Chromium Next GEM Single Cell 3’ Reagent Kit v3.1 (Cat. number 1000128, 10x Genomics, Pleasanton, CA, USA) following the manufacturer’s instructions.
Pool the libraries for sequencing.
Paired-end sequencing on the Illumina NovaSeq 6000 platform (SP Flow Cell, Illumina) with a sequencing depth of 300 million reads per library was performed by GENEWIZ GmbH (Leipzig, Germany).
scRNA-Seq data were deposited into GEO under accession number GSE274289