Apr 18, 2024

Public workspaceParse Evercode WT v2 -- University of Minnesota TMCs V.2

DOI
dx.doi.org/10.17504/protocols.io.dm6gpz8rplzp/v2
  • Laura J Niedernhofer1,
  • David A Bernlohr1
  • 1University of Minnesota, Minneapolis, MN
Allie Pybas
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gpz8rplzp/v2
Protocol Citation: Laura J Niedernhofer, David A Bernlohr 2024. Parse Evercode WT v2 -- University of Minnesota TMCs. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz8rplzp/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 18, 2024
Last Modified: April 18, 2024
Protocol Integer ID: 98428
Funders Acknowledgement:
NIH
Grant ID: 5U54AG076041-03
NIH
Grant ID: 5U54AG079754-02
Abstract
Combinatorial barcoding is an instrument-free method used to generate single-cell and single-nucleus RNA-sequencing libraries. Outlined here are the methods used to isolate single-nuclei from frozen tissue; to fix nuclei following isolation; to ligate nuclei-specific barcodes to transcript cDNA by iteratively splitting and pooling nuclei suspensions between multiple 96-well plates via the Evercode WT system; and to prepare barcoded cDNA for sequencing by synthesis (SBS). The following protocol has been adapted from protocols developed by 10x Genomics, Parse Biosciences, 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.

Single Nuclei Dissociation
Single Nuclei Dissociation
Download CG000505_Rev-A.pdfCG000505_Rev-A.pdf2.5MB

Nuclei Fixation Protocol
Nuclei Fixation Protocol
Download Evercode_Fixation_v2.1.1.pdfEvercode_Fixation_v2.1.1.pdf3.1MB

Evercode WT Protocol
Evercode WT Protocol
Download Evercode_WT_Mini_v2.2.1.pdfEvercode_WT_Mini_v2.2.1.pdf5.2MB
Note
Sequence with the read format 66,8,8,86


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.

Multiplexed FASTQ files were split by sample using a script provided by Parse Biosciences (parse_fastq_sep_groups.py, v0.4).