Oct 31, 2024

Public workspaceRNA-seq for human iPSC-CM following GSK3 inhibition

DOI
dx.doi.org/10.17504/protocols.io.kqdg32rb1v25/v1
  • 1Duke University;
  • 2Korea Advanced Institute of Science and Technology
Taylor Anglen
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DOI: dx.doi.org/10.17504/protocols.io.kqdg32rb1v25/v1
Protocol Citation: Taylor Anglen, Irene M. Kaplow, Baekgyu Choi, Kevin Hagy, Duc Tran, Magan E. Ramaker, Alejandro Barrera, Svati Shah, Inkyung Jung, Ravi Karra, Yarui Diao, Charles A. Gersbach 2024. RNA-seq for human iPSC-CM following GSK3 inhibition. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg32rb1v25/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 18, 2024
Last Modified: October 31, 2024
Protocol Integer ID: 110344
Funders Acknowledgement:
NIH
Grant ID: HG012053
Abstract
This protocol describes RNA-seq methods in human iPSC-CM following growth with or without GSK3 inhibition using CHIR99021.
Matrigel coating tissue culture plates
Matrigel coating tissue culture plates
Prepare Matrigel-coated vessels as needed, following the manufacturer’s instructions. Use coated vessels within two weeks.
Thaw Matrigel aliquot and dilute 1:30 in cold DMEM/F12.
Add Matrigel solution to plates quickly (2 ml for a 6-well, 12 ml for a 10-cm, 32 ml for a 15-cm).
Incubate at 37°C for a minimum of 1 hour. Use plates before the medium evaporates or within two weeks.
iPSC to cardiomyocyte (iPSC-CM) differentiation with WTC11 cell line
iPSC to cardiomyocyte (iPSC-CM) differentiation with WTC11 cell line
Aspirate media and add dPBS to wash. Then, aspirate dPBS. The differentiation protocol is based on previously published work. (1)
Add AccutaseTM to cells to dissociate them (1 ml for a 6-well, 3 ml for a 10-cm). Incubate at 37°C for 3 to 5 minutes.
Add 1:1 warm DMEM/F12 to the AccutaseTM and pipette gently 2 to 3 times to promote a single-cell suspension. Transfer cells to a conical tube and centrifuge at 300g for 5 minutes.
Aspirate medium and resuspend cells in mTeSR+TM (mTeSR+TM basal medium + supplement) + Rock inhibitor (10uM final concentration).
Seed cells in Matrigel coated plates quickly so that in 72hrs cells are 70% to 80% confluent. (2ml for 6-well, 12ml for a 10-cm of mTeSR+TM)
Change medium to RB- (RPMI 1640 + 50x B-27‱ supplement - insulin) + CHIR99021 (10 µM final concentration) (3 ml for a 6-well, 18 ml for a 10-cm). Volumes per tissue culture plate will remain consistent during differentiation.
After 48 hours, change medium to RB- + IWP2 (7.5 µM final concentration). Change media as close to 48 hours as possible, within +/- 5 minutes.
After another 48 hours, change medium to RB-. Change media as close to 48 hours as possible, within +/- 5 minutes.
After another 48 hours, change medium to RB+ (RPMI 1640 + 50x B-27‱ supplement with 100 U/ml penicillin and 100 µg/ml streptomycin). Change media as close to 48 hours as possible, within +/- 5 minutes.
After another 48 hours, change medium to RB+. Change media as close to 48 hours as possible, within +/- 5 minutes. Check for cell beating.
After another 48 hours, check for cell beating. If beating has not occurred, replace with RB+ until beating occurs. If beating has occurred, change medium to NG+ (RPMI 1640 - glucose + 50x B-27‱ supplement with 100 U/ml penicillin, 100 µg/ml streptomycin, 500 µg/ml recombinant human albumin, 213 ng/ml ascorbic acid, and 0.748 µl/ml 60% w/w sodium lactate solution), based on CDM3 medium. (2)
After another 48 hours, change medium to NG+.
After 48 hours, replate cells in an appropriate dish and density (3 x 10^6 cells for a 6-well, 1.8 x 10^7 cells for a 10-cm). To do this, aspirate media, add dPBS to wash, and then aspirate dPBS. Differentiation is complete at this point. Cells are now referred to as iPSC-CMs.
Add trypsin-EDTA (0.05%) to cells to dissociate them (1 ml for a 6-well, 3 ml for a 10-cm). Incubate at 37°C for 3 minutes.
Add a 1:1 mixture of stop media (DMEM + 5% FBS + DNase at 20 µg/ml final concentration) to quench the trypsin-EDTA (0.05%). Pipette gently to dislodge cells from the tissue culture plate and form a single-cell suspension, limiting to 3 to 5 repetitions. Transfer cells to a conical tube and centrifuge at 300g for 5 minutes.
Resuspend cells in RB+ + Rock inhibitor (10 µM final concentration). Seed cells in a 6-well plate over six wells (3 x 10^6 cells per well).
After 24 hours, change medium to RB+ (3 ml for a 6-well). Repeat every 48 hours for 144 hours.
Induce proliferation and developmental phenotypes in iPSC-CMs
Induce proliferation and developmental phenotypes in iPSC-CMs
Change the medium to RB+ for half the wells. For the remaining wells, change to RB+ + CHIR99021 (4 µM final concentration). Repeat every 48 hours for 168 hours.
RNA-seq
RNA-seq
iPSC-CMs were cultured for 28 days following the initiation of differentiation.
Isolate RNA using the Total RNA Purification Plus Kit (Norgen) and submit to Azenta for standard RNA-seq with ribosomal RNA depletion.
Trim reads using Trimmomatic v0.32 to remove adapters, and then align to GRCh38 using the STAR aligner v2.4.1a. (3,4)
Obtain gene counts with featureCounts from the subread package (version 1.4.6-p4) using the comprehensive gene annotation in Gencode v22. (5)
Conduct differential expression analysis with DESeq2, where gene counts are fitted to a negative binomial generalized linear model, and a Wald test identifies significantly differentially expressed genes. (6)
Protocol references
(1) Jiang, L., Yin, M., Wei, X., Liu, J., Wang, X., Niu, C., Kang, X., Xu, J., Zhou, Z., Sun, S., Wang, X., Zheng, X., Duan, S., Yao, K., Qian, R., Sun, N., Chen, A., Wang, R., Zhang, J., … Meng, D. (2015). Bach1 represses Wnt/β-catenin signaling and angiogenesis. Circulation Research, 117(4), 364–375. https://doi.org/10.1161/CIRCRESAHA.115.306829

(2) Burridge, P. W., Matsa, E., Shukla, P., Lin, Z. C., Churko, J. M., Ebert, A. D., Lan, F., Diecke, S., Huber, B., Mordwinkin, N. M., Plews, J. R., Abilez, O. J., Cui, B., Gold, J. D., & Wu, J. C. (2014). Chemically defned generation of human cardiomyocytes. Nature Methods, 11(8), 855–860. https://doi.org/10.1038/nMeth.2999

(3) Bolger, A. M., Lohse, M., & Usadel, B. (2014). Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics, 30(15), 2114–2120. https://doi.org/10.1093/bioinformatics/btu170

(4) Dobin, A., Davis, C. A., Schlesinger, F., Drenkow, J., Zaleski, C., Jha, S., Batut, P., Chaisson, M., & Gingeras, T. R. (2013). STAR: Ultrafast universal RNA-seq aligner. Bioinformatics, 29(1), 15–21. https://doi.org/10.1093/bioinformatics/bts635

(5) Liao, Y., Smyth, G. K., & Shi, W. (2014). FeatureCounts: An efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics, 30(7), 923–930. https://doi.org/10.1093/bioinformatics/btt656

(6) 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