Apr 19, 2024

Public workspace10x Protocols: Chromium Next GEM Single Cell 3ʹ -- University of Minnesota TMCs (CG000315 Rev E)

DOI
https://dx.doi.org/10.17504/protocols.io.261ge5ozdg47/v1
  • Laura J Niedernhofer1,
  • David A Bernlohr1
  • 1University of Minnesota, Minneapolis, MN
  • Cellular Senescence Network (SenNet) Method Development Community
Allie Abdellatif
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DOI: https://dx.doi.org/10.17504/protocols.io.261ge5ozdg47/v1
Protocol Citation: Laura J Niedernhofer, David A Bernlohr 2024. 10x Protocols: Chromium Next GEM Single Cell 3ʹ -- University of Minnesota TMCs (CG000315 Rev E). protocols.io https://dx.doi.org/10.17504/protocols.io.261ge5ozdg47/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: April 19, 2024
Last Modified: April 19, 2024
Protocol Integer ID: 98430
Keywords: chromium next gem single cell, chromium next gem single cell 3ʹ, 10x genomic, minnesota genomics center, barcoded cdna, cdna, sequencing, nuclei isolation, 10x protocol, cell, chromium, next gem, nuclei, specific barcode, cell dissociation
Funders Acknowledgements:
NIH
Grant ID: 5U54AG076041-03
NIH
Grant ID: 5U54AG079754-02
Abstract
Following single-cell dissociation or nuclei isolation, the Next GEM 3' assay uses microfluidics to partition and assign cell- or nuclei-specific barcodes to transcript cDNA at the 3' end. Barcoded cDNA is prepared with adaptors for sequencing by synthesis (SBS). 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.
Troubleshooting
Tissue Preparation
Complete single cell or nuclei isolation prior to starting this protocol
Library Preparation
Download CG000315_Rev-E (4).pdfCG000315_Rev-E (4).pdf4.2MB

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