Target sequences of the short hairpin RNA (shRNA) for UHRF1, DNMT1 and DNMT3B are listed in Supplementary Table 1 . The oligonucleotide containing target sequence was inserted into the AgeI/EcoRI site of the pLKO.1-TRC vector (Addgene, Watertown, MA, USA, #8453) according to standard protocols. To produce virus particles, we co-transfected the pLKO.1-TRC vector containing a specific shRNA sequence with the VSV-G envelope expressing plasmid pMD2.G (Addgene, #12259) and lentiviral packaging plasmid psPAX2 (Addgene, #12260) into HEK293T cells. Supernatants, including virus particles, were harvested 2 and 3 days after transfection and were used for viral infection with 8 µg/ml hexadimethrine bromide (Sigma-Aldrich). Twenty-four hours after infection, 1 µg/ml puromycin (Sigma-Aldrich) was used to positively select for infected cells.
Plko 1 trc vector
Manufactured by Addgene
Sourced in United States
The PLKO.1-TRC vector is a lentiviral expression vector designed for the expression of genetic elements. It contains a puromycin resistance gene for selection of transduced cells. The vector is part of the TRC (The RNAi Consortium) collection and can be used for lentiviral-mediated gene delivery applications.
Automatically generated - may contain errors
Lab products found in correlation
Pspax2, by Addgene (6 mentions)
Pmd2 g, by Addgene (5 mentions)
Puromycin, by Merck Group (4 mentions)
Fugene hd transfection reagent, by Promega (3 mentions)
Polybrene, by Merck Group (3 mentions)
Sirna sequence design software, by Takara Bio (2 mentions)
Pgl3.0 basic vector, by Promega (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Plenti c myc ddk p2a puro, by OriGene (1 mentions)
Pentr4 flag, by Addgene (1 mentions)
Plenti cmv puro dest, by Addgene (1 mentions)
Pbp fgfr2b wt, by Addgene (1 mentions)
Genejuice transfection reagent, by Merck Group (1 mentions)
Myogenin, by BD (1 mentions)
Myosin heavy chain, by Developmental Studies Hybridoma Bank (1 mentions)
Kmt1a, by Cell Signaling Technology (1 mentions)
Ha peroxidase, by Merck Group (1 mentions)
Trimethyl histone h3 lys 9, by Merck Group (1 mentions)
Trimethyl histone h3 lys27, by Merck Group (1 mentions)
Total p38, by Cell Signaling Technology (1 mentions)
43 protocols using plko 1 trc vector
1
Lentiviral Knockdown of UHRF1, DNMT1, and DNMT3B
2
Lentiviral Knockdown of m6A Regulators
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shRNAs specific to each gene used in the study are as follows: METTL3 (shMETTL3-1: 5′-GCC AAG GAA CAA TCC ATT GTT-3′, shMETTL3-2: 5′-CGT CAG TAT ATT GGG CAA GTT-3′), FTO (shFTO-1: 5′-TCA CCA AGG AGA CTG CTA TTT-3′, shFTO-2: 5′-GAT CCA AGG CAA AGA TTT ACT-3′), YTHDF1 (shYTHDF1-1: 5′-CCC GAA AGA GTT TGA GTG GAA-3′, shYTHDF1-2: 5′-CCC TAC CTG TCC AGC TAT TAC-3′), YTHDF2 (shYTHDF2-1: 5′-CCA CAG GCA AGG CCC AAT AAT-3′, shYTHDF2-2: 5′-AAG GAC GTT CCC AAT AGC CAA-3′) and YTHDF3 (shYTHDF3-1: 5′-GAT AAG TGG AAG GGC AAA TTT-3′, shYTHDF3-2: 5′-TAA GTC AAA GAA GAC GTA TTA-3′), and YTHDC1 (shYTHDC1-1: 5′-TGG ATT TGC AGG CGT GAA TTA-3′, shYTHDC1-2: 5′-CAC CAG AGA CCA GGG TAT TTA-3′). They were cloned into the pLKO.1-TRC vector (Addgene plasmid 10878, Cambridge, MA, USA) and packaged into lentiviruses according to the manufacturer's instructions. Stable knockdown cell lines were generated by lentiviral infection followed with puromycin selection. Vero cells were selected under puromycin at 10 μg/ml, while HEK293T and RD cells were at 2 μg/ml.
3
Lentiviral Overexpression and Knockdown of ONECUT2
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ONECUT2 overexpression vector pCMV6-XL5-hONECUT2 was kindly provided by Dr. Merlin Crossley. To construct lentiviral vector of pLKO.1-TRC-shONECUT2, two shRNA oligonucleotides targeting different regions of ONECUT2 were inserted to pLKO.1-TRC vector (10878, Addgene), respectively. Sequences of ONECUT2 shRNAs are listed in Supplementary Table 3 . To construct lentiviral vector of pLenti-CMV-Puro-DEST-Flag-ONECUT2, ONECUT2 protein coding DNA sequence was inserted to entry vector pENTR4-FLAG (17423, Addgene) first and then transferred to pLenti-CMV-Puro-DEST (17452, Addgene). pLenti-C-Myc-DDK-P2A-Puro-ONECUT2 (RC211951L3) and pLenti-C-Myc-DDK-P2A-Puro (PS100092) were purchased from OriGene. Lentiviral particle production and infection were performed as described previously46 (link). In brief, lentiviral vectors were co-transfected with psPAX2 and pMD2G vectors into HEK293T cells. Supernatants were collected at 24 and 48 h after transfection and stored in −80 °C. For infection, 5 × 104 cells per well were seeded in six-well plates and infected with lentiviral supernatant on the following day.
4
Generation of FGFR2-Manipulated T47D Cell Lines
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T47D shFGFR2 cell line was generated with lentiviral system based on pLKO.1-TRC vector (Addgene, #10878) [25] (link) with cloned shRNA designed on the basis of the following siRNA sequence of FGFR2 5′- GAG AUU UGG UAU UUG GUU GGU GGC - 3′ [26] (link), [27] . In all experiments with FGFR2-negative variants of cell lines as a control we used cells transfected with backbone pLKO.1 plasmid. T47D FGFR2 cells were established with retroviral vector pBp-FGFR2b-WT (Addgene, #45698) [28] (link).
5
Lentiviral Particle Generation and shRNA Cloning
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The third generation lentiviral packaging system was used for the generation of lentiviral particles as previously described [10 (link)]. In brief, HEK293T cells were transfected with the lentiviral vector of choice and three different packaging plasmids (pMDLgpRRE, pRSVREV and pMD2VSVG) using GeneJuice® Transfection Reagent (Merck Millipore). Virus supernatant was collected at time points 48 h, 72 h and 96 h post transfection. Interference RNA sequences against CHKα were designed with the software Primer3 [73 (link)] and cloned into the pLKO.1 TRC vector (Addgene plasmid #10878, [74 (link)]). Plasmids containing shRNAs against ZEB1 were derived as described previously [12 (link)].
6
Lentiviral Constructs for KMT1A and p38 Studies
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Lentiviral pLV vector expressing Flag-KMT1A [38 (link)] and LV-HA-MKK6EE and pLV-HA-MKK6DN were generated by subcloning inserts from pcDNA-HA-MKK6EE and pcDNA-HA-MKK6DN (provided by Dr. L. Puri) [39 (link)] into pLV vector. For expression of shRNA, KMT1A, p38α, or scramble shRNAs are cloned individually into lentiviral pLKO.1-TRC vector (Addgene) and sequence verified. The shRNA sequences for KMT1A and scramble were described previously [38 (link)]. The sequence for p38α shRNA was 5′-AGCCCAGCAACCTAGCTGTTT-3′. Vectors pGEX-4T-3-H3(N) [26 (link)] and pGEX-ATF2 (provided by Dr. J. Han) [40 (link)] express GST fusion N-terminal histone H3 and ATF2 proteins, respectively.
Antibodies used were phospho-p38 (Cell Signaling 9215), β-actin-peroxidase (Sigma A3854), Flag-M2 (Sigma F3165), myogenin (BD Pharmingen 556358), KMT1A (Cell Signaling 8729, and Millipore 07-550 and 05-615), MyoD (Santa Cruz sc-760 and BD Pharmingen 554130), p38α (Cell Signaling 9790), HA-peroxidase (Sigma H6533), acetyl-histone H3 (Millipore, 06-599), trimethyl-histone H3 (Lys-9) (Millipore 07-442), trimethyl-histone H3 (Lys27) (Millipore 07-449), GAPDH (Biodesign H86504M), Brg-1 (Santa Cruz sc-10768), p21cip1 (Santa Cruz sc-397), myosin heavy chain (Developmental Studies Hybridoma Bank, MF-20), total p38 (Cell Signaling 9212), and normal rabbit IgG (Santa Cruz sc-2027).
Antibodies used were phospho-p38 (Cell Signaling 9215), β-actin-peroxidase (Sigma A3854), Flag-M2 (Sigma F3165), myogenin (BD Pharmingen 556358), KMT1A (Cell Signaling 8729, and Millipore 07-550 and 05-615), MyoD (Santa Cruz sc-760 and BD Pharmingen 554130), p38α (Cell Signaling 9790), HA-peroxidase (Sigma H6533), acetyl-histone H3 (Millipore, 06-599), trimethyl-histone H3 (Lys-9) (Millipore 07-442), trimethyl-histone H3 (Lys27) (Millipore 07-449), GAPDH (Biodesign H86504M), Brg-1 (Santa Cruz sc-10768), p21cip1 (Santa Cruz sc-397), myosin heavy chain (Developmental Studies Hybridoma Bank, MF-20), total p38 (Cell Signaling 9212), and normal rabbit IgG (Santa Cruz sc-2027).
7
Establishing FGFR2 and RSK2 Knockdown Cell Lines
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MCF7/FGFR2(-) and MCF7/RSK2(-) cell lines were generated with lentiviral system based on pLKO.1-TRC vector (Addgene, #10878) with cloned shRNA designed on the basis of the following siRNA sequences: FGFR2 5′-GAG AUU UGG UAU UUG GUU GGU GGC –3′ [61 (link)], RSK2 5′-UUG CUG UCC AUU CUC AGC GCU–3′ [62 (link)]. Overexpression of RSK2 was generated with pWZL Neo Myr Flag RPSK6A3 plasmid (Addgene, #20627). Transfection was done with TurboFect Transfection Reagent (Thermo Scientific) according to the manufacturer's protocol. Stable cell line expressing constitutively active RSK2 were established by neomycin (Sigma Aldrich) selection.
8
Genetic Manipulation in Mouse Models
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The coding regions of mouse Prdm14 and Socs3 were inserted into PiggyBac transposon vectors (PB) carrying Flag or HA tag. shRNA sequences, designed to target the gene-specific regions of mouse Prdm14, Socs3 and Stat3, were cloned into pLKO.1-TRC vector (#10878, Addgene). The primer sequences used have been listed in Tables S1 and S2 .
9
Star-PAP and BIK Overexpression and Knockdown
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Star-PAP cDNA (Genecopoeia, Rockville, MD, USA) was cloned into pCDNA3.1(+) vector for transient overexpression. For Tet-On inducible expression, Star-PAP was cloned into pLVX-TRE3G response vector (Clontech, Mountain View, CA, USA), and then the lentivirus production and infection was conducted according to the manufacturer's instructions. For Star-PAP knockdown, two previously reported siRNAs were used,6 (link) and a shRNA sharing the same target sequence with siRNA#1 was used to generate stable cell line. The shRNAs for BIK were derived from TRC library database (GPP web portal, Broad Institute, Cambridge, MA, USA) and cloned into pLKO.1-TRC vector (Addgene, Cambridge, MA, USA), and lentivirus production and infection was conducted according to the TRC protocols.
10
Luciferase Assay for Odorant Receptor Promoters
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For the luciferase assay, genomic DNA was prepared and the OR1N1, OR4F6, OR7A17 and OR10G2 promoter region (−1487 to 0, −1500 to +20, −1365 to 0 and −1022 to 0, respectively) was inserted into the pGL4.12-basic vector (Promega). The OR1N1, OR4F6, OR7A17 and OR10G2 promoter sequence were amplified from human genomic DNA using primer pairs (Supplementary Table S1 ). pEGFP-G9a, pCMV-Flag-LSD1, pCMV-Suv39h1, and pCMV10-Flag-KDM3B were previously described6 (link)8 (link)62 (link). Short hairpin RNAs (shRNAs) against G9a were previously described and against LSD1 and OR10G2 were designed using the siRNA sequence designer software (Clontech)6 (link). A double-stranded oligonucleotide for shRNA plasmid construction was produced using primers from the 5′ to the 3′ end (Supplementary Table S1 ). These oligonucleotides were inserted into the AgeI/EcoRI site of the pLKO.1 TRC vector (Addgene).
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