Nov 13, 2025
Fast-ATACseq protocol – Stanford Bone Marrow TMC
- Patricia Favaro1,
- Warren Reynolds1,
- Reema Baskar1,
- Sean Bendall1
- 1Stanford University
- Human BioMolecular Atlas Program (HuBMAP) Method Development CommunityTech. support email: [email protected]
Protocol Citation: Patricia Favaro, Warren Reynolds, Reema Baskar, Sean Bendall 2025. Fast-ATACseq protocol – Stanford Bone Marrow TMC. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl464b8go5/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: November 12, 2025
Last Modified: November 17, 2025
Protocol Integer ID: 232241
Keywords: Fast-ATAC-seq, HuBMAP, Tissue Map Center (TMC), Bone Marrow, bulk atac, atacseq protocol, enriched cell, purified cell, cell suspension, atac library, dna purification, atac, mobilized peripheral blood, cell, cocktail of surface antibody, surface antibody, staining media, seq analysis, peripheral blood
Funders Acknowledgements:
HuBMAP NIH
Grant ID: U54HL165445
Abstract
Frozen CD34⁺ enriched cells from mobilized peripheral blood were thawed and suspended in cell-staining media with TruStain FC blocker for 10 min at room temperature prior to staining. Next, the cocktail of surface antibodies was added (Table S1), and cells were stained in the dark for 30 min on ice and then washed in cell-staining media. Prior to data acquisition, cell suspensions were spiked with 7-AAD (BioLegend) to label non-viable cells. The sorted populations (Figures S3B and S5D) were used for bulk ATAC-seq analysis. For bulk Fast-ATAC-seq, cells were processed as described previously (M.R. Corces et al., Nat. Genet., 48 (2016)). Briefly, approximately 50,000 FACS-purified cells were pelleted and used for the transposition reaction for each technical replicate, followed by DNA purification. Amplification and purification of the transposed fragments were performed as described previously (J.D. Buenrostro et al., Curr. Protoc. Mol. Biol., 109 (2015)) with modified indexing primers (J.D. Buenrostro et al., Nature, 523 (2015)). Libraries were quantified using qPCR. All Fast-ATAC libraries were sequenced on an Illumina NovaSeq through Novogene. Each population analyzed included two technical replicates and two biological replicates.
Troubleshooting
Details
Table S1 (novel EMP and MP FACS Panel with the cocktail of surface antibodies, Figures S3B and S5D): https://doi.org/10.1016/j.celrep.2025.115913
ATAC-seq protocol:
M.R. Corces et al., Nat. Genet., 48 (2016) - 10.1038/ng.3646
J.D. Buenrostro et al., Curr. Protoc. Mol. Biol., 109 (2015) - .1002/0471142727.mb2129s109
J.D. Buenrostro et al., Nature, 523 (2015) - 10.1038/nature14590
Acknowledgements
This work was supported by HuBMAP NIH grant U54HL165445. The study was also supported by a research grant from bluebird bio, which also provided the mPB used in this experiment.