Oct 31, 2024
HiCAR for mouse HL-1 cells following enhancer perturbations
- Taylor Anglen1,
- Irene M. Kaplow1,
- Baekgyu Choi2,
- Kevin Hagy1,
- Duc Tran1,
- Magan E. Ramaker1,
- Svati Shah1,
- Inkyung Jung2,
- Ravi Karra1,
- Yarui Diao1,
- Charles A. Gersbach1
- 1Duke University;
- 2Korea Advanced Institute of Science and Technology
Protocol Citation: Taylor Anglen, Irene M. Kaplow, Baekgyu Choi, Kevin Hagy, Duc Tran, Magan E. Ramaker, Svati Shah, Inkyung Jung, Ravi Karra, Yarui Diao, Charles A. Gersbach 2024. HiCAR for mouse HL-1 cells following enhancer perturbations. protocols.io https://dx.doi.org/10.17504/protocols.io.36wgqn2yygk5/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: October 19, 2024
Last Modified: October 31, 2024
Protocol Integer ID: 110366
Funders Acknowledgement:
NIH
Grant ID: HG012053
Abstract
This protocol describes ATAC-seq methods in human iPSC-CM following growth with or without GSK3 inhibition using CHIR99021.
Fibronectin + Gelatin coating tissue culture plates
Fibronectin + Gelatin coating tissue culture plates
5ug/ml fibronectin + 0.02% gelatin solution was added to tissue culture plates for 1 hour at 37C
Culture of HL-1 mouse atrial cardiomyocytes
Culture of HL-1 mouse atrial cardiomyocytes
HL-1 cells were culture per manufactures instructions in complete Claycomb medium (Claycomb basal medium + 10% FBS + Norepinephrine (0.1mM final concentration) + 1x Glutamax + 100 U ml−1 penicillin and 100 μg ml−1 streptomycin). (1)
Transfections for high-titer lentiviral production
Transfections for high-titer lentiviral production
Plate 1.2 x 106 or 7 x 106 HEK293T cells in a 6 well plate or 10 cm dish in the afternoon with 2 mL or 12 mL of complete opti-MEM (Opti-MEM‱ I Reduced Serum Medium supplemented with 1x Glutamax, 5% FBS, 1 mM Sodium Pyruvate, and 1x MEM Non-Essential Amino Acids). Based on previously published protocols. (2)
The next morning, transfect HEK293T cells with 0.5 μg pMD2.G, 1.5 μg psPAX2, and 0.5 μg transgene for 6 well plates or 3.25 μg pMD2.G, 9.75 μg psPAX2, and 4.3 μg transgene for 10 cm dishes using Lipofectamine 3000.
Exchanged media 6 hours after transfection and collect and pool lentiviral supernatant at 24 hours and 48 hours after transfection.
Centrifuged lentiviral supernatant at 600g for 10 min to remove cellular debris.
Concentrate lentivirus to 50–100× the initial concentration using Lenti-X Concentrator (Takara Bio). Resupsend viral pellet in dPBS
Snap freeze virus in liquid nitrogen and store at -80C
Determining viral titer
Determining viral titer
Plate 4x106 HEK293T cells per well in a 6-well plate in complete DMEM (DMEM supplemented with 1x Glutamax, 5% FBS, 1 mM Sodium Pyruvate, 1x MEM Non-Essential Amino Acids, and100 U/ml penicillin and 100 μg/ml streptomycin). Transduce individual wells add 0ul,0ul, 1ul, 5ul, 10ul, and 20ul of concentrated virus.
Change media 24 hours following transduction with complete DMEM.
Change media 24 hours following transduction with complete DMEM + blasticidin (8ug/ml final concentration). Add complete DMEM without blasticidin to one well that received no virus. This well will be the control well for MOI calculations. Change media every 48 hour until the well that received no virus but was supplemented with blasticidin has no remaining adherent cells, usually 96 hours.
Count surviving cells across all wells. Plot viral load (ul) vs % survival and generate a linear fit with the y-intercept = 0.
Transduce HL-1 cells
Transduce HL-1 cells
Transduce HL-1 with virus for a desired MOI of 0.3.
Change media with complete Claycomb medium 24 hours following transduction.
Change medium complete Claycomb medium + blasticidin (8ug/ml final concentration) 48 hours following transduction.
Change medium complete Claycomb medium + blasticidin (8ug/ml final concentration) every 48 hours for 96 hours total culture.
Change medium complete Claycomb medium every 48 hours following.
Remove stress response and fetal gene expression
Remove stress response and fetal gene expression
10 days following transduction stress stimuli was removed from culture medium for HL-1 cells. Medium was changed on transduced HL-1 with Claycomb basal medium supplemented only with 1x Glutamax and 100 U ml−1 penicillin and 100 μg ml−1 streptomycin for 72 hours.
HiCAR
HiCAR
Culture of HL-1 has occurred for 13 days following transduction, and 72 hours following the removal of stress stimuli.
Add trypsin-EDTA (0.05%) to cells to disassociate (1ml for 6-well, 3ml for a 10-cm). Incubate at 37C for 5 minutes.
Add 1:1 Claycomb medium to quench the trypsin-EDTA (0.05%). Transfer cells to a conical tube and centrifuge at 300g for 5 minutes.
Proceed to HiCAR NGS library generation as described in the original publication. (3)
HiCAR libraries were then pooled at equal molarity. Targeted enrichment was performed for regions of interest, including the areas surrounding the Myh6 transcription start site (TSS) and gene body, the Myh7 TSS, and a distal regulatory element. Biotinylated probes and protocols from Twist Bioscience were used for the targeted enrichment. (4)
Enriched libraries were sequenced using an Illumina NextSeq 1000/2000 P2 kit on an Illumina NextSeq 2000.
Sequencing data was then processed using nf-core HiCAR pipeline v1.0.0 using the mm39 genome as the reference genome.
A contact matrix was created from cooler files generated by the HiCAR pipeline for all reads mapping between mm39 chr14:55110000-55260000.
Differential contact frequency within the region was determined using DESeq2. (5)
Protocol references
(1) Claycomb, W. C., Lanson, N. A., Stallworth, B. S., Egeland, D. B., Delcarpio, J. B., Bahinski, A., & Izzo, N. J. (1998). HL-1 cells: A cardiac muscle cell line that contracts and retains phenotypic characteristics of the adult cardiomyocyte (simian virus 40 T oncogenepotassium channelsdofetilide). In Cell Biology (Vol. 95). www.pnas.org.
(2) McCutcheon, S. R., Swartz, A. M., Brown, M. C., Barrera, A., McRoberts Amador, C., Siklenka, K., Humayun, L., ter Weele, M. A., Isaacs, J. M., Reddy, T. E., Allen, A. S., Nair, S. K., Antonia, S. J., & Gersbach, C. A. (2023). Transcriptional and epigenetic regulators of human CD8+ T cell function identified through orthogonal CRISPR screens. Nature Genetics, 55(12), 2211–2223. https://doi.org/10.1038/s41588-023-01554-0
(3) Wei, X., Xiang, Y., Peters, D. T., Marius, C., Sun, T., Shan, R., Ou, J., Lin, X., Yue, F., Li, W., Southerland, K. W., & Diao, Y. (2022). HiCAR is a robust and sensitive method to analyze open-chromatin-associated genome organization. Molecular Cell, 82(6), 1225-1238.e6. https://doi.org/10.1016/j.molcel.2022.01.023
(5) Love, M. I., Huber, W., & Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12). https://doi.org/10.1186/s13059-014-0550-8