For gene knockdown experiments, JUN, TCF4 and KLF2 shRNA oligos were purchased from the Beijing Genomics Institute with target sequencing. The shRNAs were cloned into pLKO.1 vector according to the protocol, and knockdown efficiency of lentivirus was tested in P1 CMs in vitro. Then, the lentivirus was concentrated by Fast-Trap Lentivirus Purification and Concentration Kit (Millipore, FTLV00003) for mouse heart injection. Ten microliters of concentrated shNT, shJun, shTcf4, shKlf2, or shAll (Jun + Tcf4 + Klf2) lentivirus were injected into left anterior ventricles at 3 points in P1 mouse hearts, respectively. The time point of injection was chosen to allow sufficient time of viral integration, and to be sufficiently early so as to block the gradual state transition from P1 to P7. Expression of Jun, Tcf4 and Klf2 was assessed 28 days post injection. Expression of KLF2 in CMs was examined to validate inhibition of the transition state. Heart function was evaluated 28 days post injection. Afterwards, the cells from the shNT and shJun P28 mouse hearts were isolated using the Langendorff method for scRNA-seq.
Fast trap lentivirus purification and concentration kit
Manufactured by Merck Group
The Fast-Trap Lentivirus Purification and Concentration Kit is a laboratory tool designed for the purification and concentration of lentiviral particles. It utilizes a proprietary method to efficiently capture and concentrate lentiviral vectors from cell culture supernatants.
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5 protocols using fast trap lentivirus purification and concentration kit
1
Genetic Knockdown Assay for Cardiomyocyte Transition
2
Huwe1 Knockdown in C2C12 Myoblasts and Myotubes
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Huwe1 knockdown in C2C12 myoblasts and myotubes were performed using lentiviruses. Viruses were produced by transfecting pLKO.1 shRNA plasmids targeting Huwe1 (targeting sequences: #1: 5′-GCACTCTTCATAACTCACTTT-3′; #2: 5′-GCACTGCTCATCAAAGATGTT-3′)(CloneID:TRCN0000092553; TRCN0000092556; GE Dharmacon, Lafayette, CO) with packaging vectors into 293T cells using FuGENE HD (E2311, Promega). Supernatants were concentrated using Fast-Trap Lentivirus Purification and Concentration Kit (FTLV00003, Millipore). Huwe1 knockdown in myoblasts were performed in the presence of 8 µg/ml polybrene at around 70% confluency followed puromycin (A11138-03, Life technologies) selection (1 µg/ml). Extensive passages of surviving cells were avoided in order to maintain knockdown efficiency. Huwe1 knockdown in myotubes was performed by incubating lentiviral concentrates in the presence of 8 µg/ml polybrene on differentiation day 4 for two days, followed by puromycin selection for another 2 days (1 µg/ml) to eliminate uninfected myotubes.
3
Generation of Mutant HIV-1 Antisense Transcript
4
Generation of Mutant HIV-1 Antisense Transcript
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We utilized the sequence of the HIV-1 2574-nt antisense transcript reported previously (GenBank accession number JQ866626.1) (Kobayashi-Ishihara et al., 2012 (link)). The sequence was mutated at nucleotide 1526 (C > T), 1540 (C > A), and 1549 (C > A) to introduce three stops at codons 3, 7 and 10 in the open reading frame of the HIV-1 antisense protein. The mutated sequence of the ASP transcript (ASTmut) was cloned into the EcoRI and XbaI sites of the pLVX-Puro vector (Clontech). For the generation of lentiviral particles, the pLVX-ASTmut plasmid or the empty pLVX-Puro plasmid were transfected into Lenti-X 293T cells using the Lenti-X Packaging Single Shots (VSV-G) system (Clontech). After 3–4 days, culture supernatants were collected, and viral particles purified and concentrated using the Fast-Trap Lentivirus Purification and Concentration Kit (Millipore). Jurkat and Jurkat E4 cells were transduced with purified lentiviral particles overnight in complete RPMI medium containing 2 μg/ml polybrene. Cells were then selected in fresh medium containing puromycin. Stably transduced cells were maintained in medium containing 1 μg/ml puromycin.
5
Lentivirus Production and Titration
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Three plasmids generated in our previous study 12 (link) were used here. piNOS-N had an unrelated insert whose expression was controlled by the 3.2 Kb iNOS promoter. This insert had a length similar to that of the iNOS shRNA 12 (link) pCMV-shRNA and piNOS-shRNA had an iNOS shRNA whose expression was under control of the cytomegalovirus (CMV) promoter or iNOS promoter, respectively (Fig. 1 ). The shRNA was shRNA3 reported in the previous study 12 (link). These plasmids were used to transfect the 293FT cells to produce lentivirus as instructed by the protocol from Invitrogen (Carlsbad, CA). The viruses were purified and concentrated by using Fast-Trap Lentivirus Purification and Concentration kit from Millipore (Billerica, MA). All viruses were titrated by using Lentiviral titer kit from MellGen Laboratories Inc. (Surrey, BC, Canada). The titer of each viral solution was about 1010 transducing units/ml in phosphate buffered saline. The virus was then aliquoted and kept at -800C until used.
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