Mar 26, 2025

Public workspace10x Protocols: Chromium Next GEM Single Cell 5' -- University of Minnesota TMCs (CG000331 Rev F) V.2

DOI
dx.doi.org/10.17504/protocols.io.5qpvokn89l4o/v2
  • Laura J Niedernhofer1,
  • David A Bernlohr1
  • 1University of Minnesota, Minneapolis, MN
  • Cellular Senescence Network (SenNet) Method Development Community
Allie Pybas
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DOI: dx.doi.org/10.17504/protocols.io.5qpvokn89l4o/v2
Protocol Citation: Laura J Niedernhofer, David A Bernlohr 2025. 10x Protocols: Chromium Next GEM Single Cell 5' -- University of Minnesota TMCs (CG000331 Rev F). protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvokn89l4o/v2Version created by Allie Pybas
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: April 02, 2024
Last Modified: March 26, 2025
Protocol Integer ID: 125027
Funders Acknowledgements:
NIH
Grant ID: 5U54AG076041-03
NIH
Grant ID: 5U54AG079754-02
Abstract
 Following single-cell dissociation or nuclei isolation, the Next GEM 5' assay uses microfluidics to partition and assign cell- or nuclei-specific barcodes to transcript cDNA at the 5' end. Barcoded cDNA is prepared with adaptors for sequencing by synthesis (SBS). The following protocol has been used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. The following protocol has been adapted from protocols developed by 10x Genomics and Illumina to be used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. These protocols are owned by their respective companies and are subject to periodic revision.

Tissue Preparation
Tissue Preparation
Complete single cell or nuclei isolation prior to starting this protocol
Library Preparation
Library Preparation
Download CG000331_Chromium_Next_GEM_Single_Cell_5_v2_UserGuide_RevF.pdfCG000331_Chromium_Next_GEM_Single_Cell_5_v2_UserGuide_RevF.pdf4.7MB
Note
Sequence with the read format 29,10,10,89


Note
Sequencers used at UMN Genomics Center:
  • Illumina NextSeq 2000
  • Illumina NovaSeq 6000
  • Illumina NovaSeq X Plus


FASTQ Generation
FASTQ Generation
BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0