The shRNA plasmids were generated by cloning oligonucleotide from TRC library (Supplementary Table S1 ) into an inducible shRNA vector system, created through modifying pRSITEP-U6Tet-(sh)-EF1-TetRep-2A-Puro plasmid (Cellecta, Inc., Mountain View, CA, USA) by replacing the TetRep-2A-Puromycin site with either TetRep-P2A-Puro-P2A-RFP670 or TetRep-P2A-Puro-P2A-GFP. The vector contains a 1.2 kb stuffer sequence that was double–digested by FastDigest BshTI and EcoRI (FD1464 and FD0274; Thermo Fisher, Waltham, MA, USA) for 1 h. The 8 kb BshTI/EcoRI band was extracted from 1% agarose gel using Wizard SV Gel and PCR Clean-Up System (A9280; Promega, Madison, WI, USA). Separate forward and reverse oligos (custom DNA oligo service; Sigma) were annealed in the annealing buffer (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl and 1 mM EDTA) using a thermal cycler (C1000; Bio-Rad), incubating for 5 min at 95 °C and then cooling gradually to 20 °C, and 1 μl of annealed oligo pairs was ligated into 50–100 ng of the digested vector using T4 DNA ligase (EL0014; Thermo Fisher) and T4 Polynucleotide Kinase (EK0031; Thermo Fisher), incubated overnight at 37 °C. Transformation was conducted using One Shot Stbl3 Chemically Competent Cells (C737303; Thermo Fisher) according to the manufacturer's protocol. Next, at least three colonies were picked for colony PCR using DreamTaq Green PCR Master Mix (K1081; Thermo Fisher). The primers used for this reaction were U6-tet-F: 5′-GGA CTA TCA TAT GCT TAC CGT AAC-3′ and U6-tet-R: 5′-TGG ATG AAT ACT GCC ATT TGT CTC-3′. Colonies lacking the stuffer were sent for sequencing (à la Carte Sequencing Service; Eurofins Genomics, Ebersberg, Germany). After sequencing validation of the constructs, a GFP reporter from pRSITEP-V2-GFP vector replaced the RFP670 cassette using double-digest reactions with FastDigest SalI and FastDigest XbaI (FD0644 and FD0684; Thermo Fisher).
The γ3-hMYH overexpression plasmid was generated by PCR amplification of MYH cDNA (GenBank accession number AF527839.1) using the following primers: 5′-TAT AGT CGA CAT GAG GAA GCC ACG-3′ and 5′-TAT AGC GGC CGC TCA CTG GGC TGC ACT G-3′. Double digestion reactions were carried out with FastDigest SalI and NotI (FD0644 and FD0593; Thermo Fisher) for 1 h. The PCR product was then ligated into pENTR1A no ccDB (17398; Addgene, Cambridge, MA, USA) entry vector using T4 DNA ligase (EL0014; Thermo Fisher). The entry vectors carrying γ3-hMYH insert were verified by sequencing (Eurofins Genomics). Next, the insert from entry vector was transferred to pINDUCER20 (44012; Addgene) destination vector using Gateway LR Clonase II (11791100; Thermo Fisher). Finally, the constructs were validated by colony PCR using DreamTaq Green PCR Master Mix (K1081; Thermo Fisher).
The γ3-hMYH overexpression plasmid was generated by PCR amplification of MYH cDNA (GenBank accession number AF527839.1) using the following primers: 5′-TAT AGT CGA CAT GAG GAA GCC ACG-3′ and 5′-TAT AGC GGC CGC TCA CTG GGC TGC ACT G-3′. Double digestion reactions were carried out with FastDigest SalI and NotI (FD0644 and FD0593; Thermo Fisher) for 1 h. The PCR product was then ligated into pENTR1A no ccDB (17398; Addgene, Cambridge, MA, USA) entry vector using T4 DNA ligase (EL0014; Thermo Fisher). The entry vectors carrying γ3-hMYH insert were verified by sequencing (Eurofins Genomics). Next, the insert from entry vector was transferred to pINDUCER20 (44012; Addgene) destination vector using Gateway LR Clonase II (11791100; Thermo Fisher). Finally, the constructs were validated by colony PCR using DreamTaq Green PCR Master Mix (K1081; Thermo Fisher).
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