Jan 28, 2026
Target enrichment of 10X Genomics scRNA-seq gene expression libraries
- Boxun Li1,
- Charles Gersbach1
- 1Duke University
- Gersbach Lab
Protocol Citation: Boxun Li, Charles Gersbach 2026. Target enrichment of 10X Genomics scRNA-seq gene expression libraries. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo1qpbg4o/v1
Manuscript citation:
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: January 26, 2026
Last Modified: January 28, 2026
Protocol Integer ID: 241588
Keywords: 10X Genomics, scRNA-seq, neuron, astrocyte, target enrichment, target enrichment of 10x genomics scrna, enrichment of 10x genomics scrna, seq gene expression libraries of neuron mono, 10x genomics scrna, seq gene expression library, seq gene expression libraries this protocol, 10x genomic, mouse primary astrocyte, primary astrocyte, neuron mono
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol is adapted from the work of Boxun Li in the Gersbach lab at the Duke University.
Abstract
This protocol describes target enrichment of 10X Genomics scRNA-seq gene expression libraries of neuron mono-culture or co-culture with mouse primary astrocytes.
Troubleshooting
Target enrichment of 10X Genomics scRNA-seq gene expression libraries
Enrichment DNA probe panel for target enrichment for scRNA-seq gene expression libraries using a hybridization-based method (Twist Bioscience, PN 101001, 104445, 100856, and 101262) was designed by Twist Bioscience for the 5’ UTR and first ~400bp of the coding sequence of all transcripts annotated in Gencode v41 of the 222 transcription factor genes of interest along with 4 positive control genes (TFRC, UBQLN2, GRN, and CDH2) as well as 7 housekeeping genes (DPM1, KRIT1, BAD, ARF5, POLDIP2, HCCS, TSR3).
Target enrichment is performed on the 10X Genomics 5’ HT v2 gene expression sequencing libraries of the putative regulatory element (pRE) Perturb-seq screens, according to the manufacturer’s recommended protocol Standard Hybridization v2 Protocol (Twist Bioscience, DOC-001273 REV 3.0)
The enriched libraries were amplified using the 10X Genomics Amp Mix (10X Genomics, PN 2000047) and purified using Twist Purification Beads.
The target-enriched gene expression libraries were sequenced using Illumina Nextseq 2000, NovaSeq 6000 or NovaSeq X Plus sequencing to target depth of ~5 x 10^3 reads/cell.