Feb 25, 2026

Public workspace10x Multiome Gene Expression Library Construction and Sequencing

DOI
https://dx.doi.org/10.17504/protocols.io.dm6gp1q28gzp/v1
  • Scott Lindsay-Hewett1,
  • Tyler Ostrander1,
  • Abbas Hakim1,
  • Morgan Meads1,
  • Valentina Stanley1,
  • Mana Parast1,
  • Louise C. Laurent1
  • 1University of California San Diego
  • Human BioMolecular Atlas Program (HuBMAP) Method Development Community
    Tech. support email: [email protected]
Scott Lindsay-Hewett
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DOI: https://dx.doi.org/10.17504/protocols.io.dm6gp1q28gzp/v1
Protocol Citation: Scott Lindsay-Hewett, Tyler Ostrander, Abbas Hakim, Morgan Meads, Valentina Stanley, Mana Parast, Louise C. Laurent 2026. 10x Multiome Gene Expression Library Construction and Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp1q28gzp/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: February 24, 2026
Last Modified: February 25, 2026
Protocol Integer ID: 243985
Keywords: Multiome, 10x Genomics, single nucleus, placenta, snRNAseq, 10x multiome gene expression library construction, 10x multiome gene expression, library quantification via agilent bioanalyzer, gex, agilent bioanalyzer, pairs per nucleus, cell ranger arc
Funders Acknowledgements:
National Institutes Of Health - Female Reproductive Tissue Mapping Center
Grant ID: U54 HD104393
National Institutes Of Health - Pregnant Female Reproductive Tissue Mapping Center
Grant ID: U54 HD110347
Abstract
This protocol describes the construction, quality control, and sequencing of 10x Multiome Gene Expression (GEX) libraries. It details library quantification via Agilent Bioanalyzer, multiplexed sequencing on the Illumina NovaSeq X Plus, and initial data processing using Cell Ranger ARC to ensure a target depth of 20,000 read pairs per nucleus.
Materials
Equipment

Vortex
Picofuge
Plate spinner
Thermal Cycler (BioRad cat # C1000 Touch with 96–Deep Well Reaction Module); others okay
Qubit fluorometer (or similar device for library quantification)
Agilent Bioanalyzer (or similar capillary electrophoresis device for nucleic acid analysis)
Illumina NovaSeq X Plus (other Illumina NGS instruments okay)

Materials/reagents

Rainin pipettes (single and multichannel) and filter tips (10x approved, emulsion-safe)
DNA LoBind Tubes, 1.5 ml (Eppendorf cat # 022431021)
15 ml and 50 ml centrifuge tubes
Reagent reservoirs
TempAssure PCR 8-tube strip  (USA Scientific cat # 1402-4700)
Nuclease-free Water (Fisher cat # AM9937)
200-proof ethanol
Qiagen Buffer EB (Qiagen cat # 19086)
SPRIselect Reagent Kit (Beckman Coulter cat # B23318)
Magnetic Separator/Magnetic Separator B (10x Genomics cat # 230003/2001212)
Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle, 16 rxns (10x Genomics cat # PN-1000283)
Dual Index Kit TT Set A, 96 rxns (10x Genomics cat # PN-1000215)
Agilent DNA High Sensitivity Kit (Agilent cat # 5067-4626)
Qubit dsDNA High Sensitivity (HS) Assay Kit (Fisher cat # Q32851)
Illumina Sequencing Reagent Kit
Troubleshooting
Gene Expression (GEX) Library Construction
Construct libraries in accordance with the 10x Genomics Chromium Next GEM Single Cell Multiome ATAC + Gene Expression User Guide (CG000338). Download CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdfCG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevG.pdf

6 cycles of cDNA amplification are performed for an expected target recovery of 10,000 nuclei.

  • cDNA Quality Control (QC): Assess cDNA quality and size distribution using the Agilent Bioanalyzer High Sensitivity DNA assay.
  • cDNA Quantification: Determine cDNA concentration by selecting the region between 200–9000 bp.

Carry forward 25% total cDNA yield to Gene Expression (GEX) Library Construction, and tailor the number of sample index PCR cycles to the cDNA input, according to the User Guide.

  • GEX Library Quality Control (QC): Assess library quality and size distribution using the Agilent Bioanalyzer High Sensitivity DNA assay.
  • GEX Library Quantification: To determine library molarity, calculate the average fragment size by selecting the region between 175–1000 bp.
  • GEX Library Pooling: Prepare an equimolar pool of libraries for multiplexed sequencing and determine pool concentration using the Qubit dsDNA High Sensitivity (HS) assay.

Expected result

Typical Bioanalyzer cDNA trace.


Expected result

Typical Bioanalyzer Gene Expression Library trace.


Sequencing
Target Depth: Sequence GEX libraries to a minimum depth of 20,000 read pairs per nucleus.

Sample Sheet Generation: Populate the Illumina SampleSheet.csv using the specific index sequences from the Dual Index Kit TT Set A. Download Dual_Index_Kit_TT_Set_A.csvDual_Index_Kit_TT_Set_A.csv

NovaSeq X Plus Configuration: Use the following cycling parameters:
  • Read 1: 28 cycles
  • i7 Index: 10 cycles
  • i5 Index: 10 cycles
  • Read 2: 90 cycles
Data Pre-processing
  • Primary Analysis: Process raw FASTQ files from paired GEX and ATAC libraries using the Cell Ranger ARC pipeline to perform demultiplexing and initial cell calling.
  • Depth Normalization: Evaluate the actual GEX reads per nucleus for each sample. If the target of 20,000 GEX reads is not met, rebalance the GEX library pool and perform supplemental sequencing as required.
  • Downstream Analysis: Proceed to integrated multiomic analysis once target depth and cell recovery metrics are validated.