112 Chapter 5 reference genome GRCh37 using STAR v2.4.2a. Picard’s AddOrReplaceReadGroups v1. (http:// broadinstitute.github.io/picard/ (accessed on 4 June 2020)) was used to add read groups to the BAM files, which were sorted with Sambamba v0.4.5 and transcript abundances were quantified with HTSeq-count v0.6.1p1 using the union mode. Subsequently, reads per kilobase per million mapped reads (RPKMs) were calculated with edgeR’s RPKM function. Transfection of hiPSC-CMs with mCherry. hiPSC-CMs were transfected (ViaFect (6:1), Promega, Madison, WI, USA) with 100 ng/cm2 PF256-H2B-mCherry and 20% FBS (Gibco). After 3 days, cells were harvested for FACS analysis, and fluorescent images were acquired with the EVOS (Life Technologies, Carlsbad, CA, USA). For quantification, nuclear expression (mCherry) was calculated as a percentage of positive nucleus to total nucleus (Hoechst 33342, Invitrogen, Waltham, MA, USA). The time-lapse imaging was developed with a 12 h post-transfection of hiPSC-CMs with mCherry plasmid on an LIPSI Ti2 inverted microscope system (Nikon, Tokio, Japan) using wide-field mode or confocal mode with a 10×/0.45 NA air objective. Images were taken every 5 min for 12 h. FACS Analysis of Transfection Efficiency. After transfection, cells were dissociated via digestion with TrypLE™ Select (10×, Gibco, Waltham, MA, USA) and fixed in 4% paraformaldehyde (PFA). Next, immunostaining was performed on cells fixed with 4% paraformaldehyde (PFA) in PBS. Cells were incubated with anti-α-Actinin (clone A7811, Sigma Aldrich, Saint Louis, MO, USA) or anti-Ki67 (clone A833, Abcam, Cambridge, UK) labeled with Alexa-488 using 1:300 Alexa Fluor 488 (Thermo Fischer Scientific, Waltham, MA, USA) and 0.1 µg/mL Hoechst (33342, Invitrogen, Waltham, MA, USA). The staining was performed in PBS with 5% BSA (Roche) and 0.3% Triton-x-100 (Sigma Aldrich, Saint Louis, MO, USA). Stained cells were analyzed and sorted on an Astrios FACS (BD Pharmingen, San Diego, CA, USA). Data were collected from at least 10,000 events. Statistical analysis. Statistical analysis was conducted by Graphpad Prism software version 9. Data significance was statistically determined using an unpaired Student’s t-test for the comparison of two normally distributed datasets. One-way ANOVA or two-way ANOVA was performed with a Tukey’s multiple comparisons test to evaluate statistical differences among multiple datasets. Data plots are displayed as mean ± standard error of mean (sem), unless specified otherwise, and a p-value < 0.05 was set to determine statistical significance.
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