Jan 26, 2026

Public workspaceK562 culture and characterization

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo1krbg4o/v1
  • Lexi Bounds1,
  • Alejandro Barrera1,
  • Maria ter Weele1,
  • Siyan Liu1,
  • Euphy Wu2,
  • Shengyu Li1,
  • Revathy Venukuttan1,
  • Ruhi Rai1,
  • Wancen Mu2,
  • Nahid Iglesias1,
  • Paola Giusti-Rodriguez3,
  • Timothy Reddy1,
  • Yun Li2,
  • Reluca Gordan4,
  • Andrew Allen1,
  • Michael Love2,
  • Patrick Sullivan2,
  • Greg Crawford1,
  • Charles Gersbach1
  • 1Duke University;
  • 2University of North Carolina;
  • 3University of Florida;
  • 4University of Massachusetts
  • Gersbach Lab
Andrea R Daniel
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo1krbg4o/v1
Protocol Citation: Lexi Bounds, Alejandro Barrera, Maria ter Weele, Siyan Liu, Euphy Wu, Shengyu Li, Revathy Venukuttan, Ruhi Rai, Wancen Mu, Nahid Iglesias, Paola Giusti-Rodriguez, Timothy Reddy, Yun Li, Reluca Gordan, Andrew Allen, Michael Love, Patrick Sullivan, Greg Crawford, Charles Gersbach 2026. K562 culture and characterization. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo1krbg4o/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: January 22, 2026
Last Modified: January 26, 2026
Protocol Integer ID: 239164
Keywords: K562 cells, dCas9-KRAB, k562 cell culture, methods for k562 cell culture, k562 culture
Funders Acknowledgements:
NIH
Grant ID: HG012053
Disclaimer
This protocol was adapted from the work of Lexi Bounds in the Gersbach lab at Duke University.
Abstract
This protocol describes methods for K562 cell culture and characterization.
Materials
dCas9-KRAB (Addgene #83890)
hygromycin B (ThermoFisher #10687010)
Blasticidin S HCl (ThermoFisher #A1113903)
Invitrogen‱ SuperScript‱ VILO‱ cDNA Synthesis Kit (ThermoFisher #11754050)
PerfeCTa SYBR Green FastMix (Quantbio #95072)
Troubleshooting
K562 culture
K562 cells are obtained from the American Tissue Collection Center (ATCC) via the Duke University Cancer Center Facilities.
K562 cells are cultured in RPMI1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin.
The polyclonal dCas9-KRAB cell line are generated as previously described (ref 1). K562 cells are transduced with dCas9-KRAB (Addgene #83890) lentivirus with polybrene at a concentration of 8 ug/mL. At two days post-transduction, cells are selected for 10 days with 600 ug/mL hygromycin B (ThermoFisher #10687010). Following selection, polyclonal cells are stained to confirm expression of dCas9-KRAB protein.
K562 Characterization
dCas9-KRAB K562 cells are transduced with an individual gRNA targeting the HS2 enhancer region (ref 2) or transduced with a nontargeting gRNA (N=3 each) via spinfection. 25,000 cells are resuspended in 0.5 mL of media containing 25 uL of lentivirus and 8 ug/mL polybrene, then centrifuged for 30 minutes at 25C
The volume is transferred into one well of a 24-well plate.
24 hours post-transduction, the cells are centrifuged for 5 minutes at 300g, resuspended in fresh media, and plated into a 6-well plate.
Cells are then selected with 2.5ug/mL Blasticidin S HCl (ThermoFisher #A1113903) for 3 days.
The cells are harvested and mRNA was purified using the Qiagen RNeasy kit according to the manufacturer’s protocol.
250 ng mRNA is used as input for cDNA amplification using the Invitrogen‱ SuperScript‱ VILO‱ cDNA Synthesis Kit (ThermoFisher #11754050).
For RT-qPCR, each reaction contains 2 uL cDNA, 6 uL H20, 1 uL 10 uM FWD primer, 1 uL 10uM RVS primer, and 10 uL PerfeCTa SYBR Green FastMix (Quantbio #95072).
Delta delta Ct analysis is performed in Microsoft Excel. Graphpad Prism is utilized to conduct one-way ANOVA tests followed by Tukey’s HSD for post-hoc testing.
Protocol references
1. Klann, T. S. et al. Genome-wide annotation of gene regulatory elements linked to cell fitness. 2021.03.08.434470 Preprint at https://doi.org/10.1101/2021.03.08.434470 (2021).

2. Thakore, P. I. et al. Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nat. Methods 12, 1143–1149 (2015).