10x Genomics Library Prep

A: You have three options to create compatible 10x Genomics libraries with the Singular Genomics G4 Sequencing Platform:

1. Use Singular Genomics (SG) indexed or non-indexed t-primers during library preparation instead of the primers provided by 10x Genomics during the sample index PCR step.
2. Adapt fully constructed existing 10x Genomics libraries using SG indexed or non-indexed PCR primers.
3. Use the Max Read Kit to adapt 10x Genomics libraries.

These three options are detailed below.

(1) Using SG Primers During Library Prep

Use SG Dual Indexed t-Primers or Non-indexed t-Primers during library preparation instead of primers provided by the kit during the sample index PCR step. This approach has been validated for Chromium 3' Gene Expression library preparation protocol.

For more information, see the product overviews:

• SG t-UDI Primers Overview
• SG Non-Indexed Library Prep t-Primers Overview
• 10x FAQ Page

(2) Adapting fully constructed existing 10X Genomics libraries using Singular Genomics PCR primers.

Singular Genomics uses SP1 and SP2 sequencing primers to prime the sequencing reaction. The following 10x assays contain binding sites for these sequencing primers:

• Chromium Single Cell Gene Expression (e. g. 3' v3.1): Gene Expression libraries only
• Spatial Gene Expression for Fresh Frozen (Visium for Fresh Frozen)
• Chromium Single Cell Immune Profiling (e. g. 5' v2): Gene Expression, V(D)J, and 5' CRISPR libraries
• Single Cell Multiome ATAC + Gene Expression: Gene Expression libraries only

These libraries do still require the S1 and S2 flow cell binding sequences to be compatible with the G4. There are two protocols for incorporating these sequences:

• Guidelines to adapt libraries and add indexes: https://singulargenomics.com/wp-content/uploads/2023/06/Adapting-Library-Insert-600024.pdf
• Guidelines to adapt libraries retaining original indices: https://singulargenomics.com/wp-content/uploads/2023/06/Adapting-Library-Retain-Index-600025.pdf

(3) Using A Max Read Kit

A Max Read Kit generates multiple library fractions of one 10x Genomics library with different sequencing primer binding sites. After combining the fractions, the combined library is sequenced successively with distinct fraction-specific sequencing primers on one G4 flow cell, which enables higher output of short reads without compromising quality.

The following Max Read Kits from Singular Genomics are available:

• Max Read Single Cell and Spatial Gene Expression
• Max Read Spatial Gene Expression for FFPE

These kits support the following 10x Genomics library preparation kits:

For more information, see the Max Reads page on the Singular Genomics website.

DEMONSTRATED G4 SEQUENCING EXPERIMENTS

For a demonstrated 10x Genomics single-cell sequencing experiment on the G4, see the App Note Single Cell RNA Sequencing on the G4 .

For a demonstrated 10x Genomics single-cell sequencing experiment on the G4 using the Max Read Kit, see the poster The Max Read Kit for High-Throughput Single Cell Sequencing on the G4 .

ORDERING INFORMATION

For ordering information for 10x Genomics library preparation kits, see 10xgenomics.com .

Indexed or non-indexed SG PCR t-primers from Singular Genomics:

Indexed or non-indexed SG PCR primers from Singular Genomics:

Max Read Kit from Singular Genomics:

MORE INFORMATION

For more information, see also the 10x Genomics FAQ Can I sequence my libraries with Singular Genomics?