Genomic DNA from wild-type fission yeast cells was used as a template for the PCR amplification of N-terminally truncated CENP-A gene copies using Phusion polymerase (Thermo Fisher Scientific, Waltham, MA, USA), whereas plasmids pFA6a-6xGLY-3xFLAG-kanMX6 [49 (link)] and pFA6a-GFP(S65T)-kanMX6 [50 (link)] were used as templates [50 (link)] for FLAG and GFP protein tags, respectively. All PCR products were ethanol-precipitated and subjected to gel purification using a DNA gel extraction kit (Qiagen) according to manufacturer’s instructions. Both plasmid pREP41 and purified DNA (PCR products) were treated with restriction enzymes (New England Biolabs (NEB), Ipswich, MA, USA) and subjected to agarose gel purification, as described previously [28 (link)]. Digested gene inserts were ligated to pREP41 plasmids with T4 ligase (NEB), and transformed into TOP10 competent cells (Thermo-Fisher Scientific), before plating onto LB + Car agar plates (Luria–Bertani medium with 100 μg/mL carbenicillin (GoldBio, St. Louis, MO, USA). Positive transformants were then expanded by plasmid miniprep (Qiagen, Hilden, Germany) and extracted plasmids were sequenced using the Sanger method for confirmation of the correct gene sequences, frame, and orientation.
Top10 competent cell
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
TOP10 competent cells are a strain of Escherichia coli (E. coli) bacteria that have been genetically modified to be highly competent for efficient transformation with plasmid DNA. They are designed for high-efficiency cloning and plasmid propagation.
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Lab products found in correlation
Smarter race cdna amplification kit, by Takara Bio (4 mentions)
Spin miniprep kit, by Qiagen (3 mentions)
Pcr advantage kit, by Takara Bio (3 mentions)
Gel extraction kit, by Qiagen (3 mentions)
Qiaprep spin miniprep kit, by Qiagen (3 mentions)
T4 dna ligase, by New England Biolabs (3 mentions)
Rnaeasy mini kit, by Qiagen (2 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Dpni, by New England Biolabs (2 mentions)
Pcr purification kit, by Qiagen (2 mentions)
Hispeed plasmid maxi kit, by Qiagen (2 mentions)
Pcmv6 ac gfp, by OriGene (2 mentions)
Rfx4 v1, by OriGene (2 mentions)
Rfx4 v3, by OriGene (2 mentions)
Rfx4 v4, by OriGene (2 mentions)
Endofree plasmid maxi kit, by Qiagen (2 mentions)
Pcdna3, by Thermo Fisher Scientific (2 mentions)
Carbenicillin, by GoldBio (1 mentions)
T4 ligase, by Thermo Fisher Scientific (1 mentions)
Dna gel extraction kit, by Qiagen (1 mentions)
56 protocols using top10 competent cell
1
Cloning and Validation of Truncated CENP-A
2
Receptor Constructs for Oxytocin and AVP
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The receptor constructs were ordered from GeneScript and subcloned into a pcDNA3.1/CT-GFP-TOPO vector. The coding sequences of the GenBank entries NM_001199370.1 (oxtr), NM_001199369.1 (oxtrl), NM_001301114.1 (avpr1aa), and NM_001297676.1 (avpr1ab) were used for synthesis. The stop codons were removed, and an additional ATP was added to facilitate hybrid protein expression with GFP in frame from the vector. The GCCACC Kozak sequence was inserted before the start codons. After transformation of the plasmid constructs into TOP10 competent cells (Thermo Fisher Scientific), the Maxiprep (Thermo Fisher Scientific) method was used to amplify the plasmids for transfection.
3
Generation of AR and AR-V7 Lentiviral Constructs
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DH5a and TOP10 competent cells were purchased from Thermo Fisher Scientific, Waltham, MD and used for plasmid transformation and propagation as per manufacturer’s instructions. PCR amplified cDNA of AR (full length) was cloned from an available pCR3.1 expression vector50 (link) by fusing with 3X FLAG sequences at the beginning and an HA-Tag at the end. This was cloned into the pHAGE lentiviral expression vector as previously described25 (link). pCR3.1 AR-V7 has been described previously51 (link). The pLX302_FOXA1-V5 plasmid was purchased from Addgene, Watertown, MA.
4
TCR Sequencing from mRNA Isolation
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Briefly, the TCR was sequenced by isolating mRNA using RNAeasy Mini Kit (Qiagen, Germany) and synthesized cDNA using SMARTer RACE cDNA Amplification kit (Takara), PCR amplified using PCR Advantage Kit (Takara), and run on a 1% agarose gel for PCR band confirmation. The PCR product was then purified and transformed with TOP10 competent cells (Thermo Fisher, UK) and plated on Luria broth (LB) agar media; and colony PCR was performed to amplify product before isolating plasmid DNA using Spin Miniprep Kit (Qiagen, Germany). The resulting purified plasmid DNA was sent for sequencing at 100 nM of concentration.
5
Cloning and Sequencing of ZmLOX5 Gene
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Genomic DNA was extracted from Yu796 as described above. PCR reactions were carried out using Phusion™ High-Fidelity DNA Polymerase (Thermo Fisher Scientific™, Houston, TX, USA) and ZmLOX5-specific forward and reverse primers (listed in Table S1 ). The PCR product was loaded and separated in 1.5 % (w/v) agarose gel and purified using a gel extraction kit (QIAGEN, Germantown, MA, USA). “A-Tailing” was added to 3′ blunt-ends of 100 ng of purified PCR product using GoTaq™ DNA Polymerase (Promega, Madison, WI, USA) for 10 min at 72 °C. The gel-purified DNA fragments were cloned into a TA-vector using the TOPO™ TA Cloning™ Kit (Thermo Fisher Scientific™, Houston, TX, USA), and subsequently, the resulting plasmid constructs were transformed into TOP10 competent cells (Thermo Fisher Scientific™, Houston, TX, USA). The plasmids were extracted from the TOP10 E. coli strains positive for ZmLOX5 fragment insertions and purified for the next step sequencing.
6
Lentiviral and Adenoviral Knockdown of CREB3L2
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SureSilencing short hairpin (sh) plasmids for knocking down CREB3L2 were purchased from Qiagen and subcloned into pRRL-PGK lentiviral transfer vector for transformation of TOP10 competent cells (Thermo Fisher). After maxi-prepping, plasmids corresponding to Creb3l2 sh2 and sh4, as well as GFP and scrambled (scr) controls, were transfected along with packaging plasmids into HEK293FT cells (Thermo Fisher) by Ca-PO4-DNA precipitation. Viral particles were harvested and concentrated by centrifugation using Amicon 0.45 mm filters (Sigma Aldrich). For adenoviruses, a plasmid containing a U6 promoter-driven Creb3L2 shRNA targeting mouse sequence in an adenoviral backbone co-expressing mCherry was obtained from Vector Builder. Non-targeting control (shSAFE) was provided by the University of Iowa Viral Vector Core Facility. AdRIP-proCpepRUSH has been described elsewhere [32 (link)]. Recombinant adenoviruses were generated in HEK293 cells and purified by cesium chloride gradient. All sequences were verified by the Iowa Institute of Human Genetics, University of Iowa.
7
Cloning of E. coli LPS Biosynthesis Genes
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All primers used are listed in the supplement (S3 Table ). PCR was performed using Phusion High-Fidelity DNA Polymerase (New England BioLabs). Suspension of colonies of the E. coli wild type strain, BW25113[33 (link)] or MG1655 (ATCC 47076), was used as the template. Plasmid DNA and PCR fragments were purified using the Qiaprep spin miniprep kit (Qiagen) or the Qiaquick PCR purification kit (Qiagen), respectively. The E. coli lpxA, lpxD, and lpxK genes were PCR-amplified using primers: TU242 and TU243 for lpxA, TU115 and TU116 for lpxD, and MM18 and MM19 for lpxK. The PCR product was digested with EcoRI and HindIII, purified, and cloned into the EcoRI and HindIII sites of pMMB206[34 (link)]. Ligation was performed using Quick Ligase (NEB) or Takara Ligation Kit (Takara). DH5α or Top10 competent cells (Thermo Fisher Scientific) were used for transformation followed by selection on L agar supplemented with 10 μg/mL chloramphenicol. The DNA sequences of the inserts were confirmed by nucleotide sequencing by Quintara Biosciences using primers TU40 and TU96. The generated plasmids were designated as pTU457 [pMMB206 Plac::lpxA], pTU433 [pMMB206 Plac::lpxD], and pMM14 [pMMB206 Plac::lpxK].
8
Sequencing T-Cell Receptor CDR3 Regions
9
TCR-β Repertoire Analysis of CD8+ T Cells
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One million cells from each epitope-specific polyclonal CD8+ T-cell line were harvested and washed three times with Phosphate Buffered Saline. Total RNA was extracted using the RNeasy Plus Mini kit (Qiagen, Germany), and cDNA was then synthesized from 300 ng RNA using the SMARTer RACE cDNA amplification kit (Takara Bio, Japan) following the manufacturer's instruction. Subsequently, cDNA was amplified for variable regions of the TCR-β chain using the PCR Advantage kit (Takara Bio), with the primer 5′-TGCTTCTGATGGCTCAAACACAGCGACCT-3′ and run on a 1.2% agarose gel for PCR band confirmation (at 500 bp). PCR products were purified using the Monarch DNA Gel Extraction kit (New England BioLabs, USA) and then transformed into TOP10 competent cells (ThermoFisher). Plasmid DNA was extract using the Spin Miniprep kit (Qiagen) followed by Sanger sequencing.
10
Introducing Variants into ACTG1 Plasmid
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The novel mutations c.102 C > G, c.110 G > A, c.246 G > A, c.493 C > G c.823 C > T, and c.994 C > T, and the previously reported variants c.142 G > C, c.266 C > T, c.353 A > T, c.354 G > C, c.721 G > A, c.791 C > T, c.895 C > G and c.914 T > C were introduced into the ACTG1 plasmid (pFN21A HaloTag CMV ACTG1 plasmid: purchased from Kazusa DNA Research Lab, FHC07847) according to manufacturer’s protocol using an In-Fusion HD Cloning Plus Kit (Takara Bio, Shiga, Japan). All variants mentioned in this study were shown in NM_001614.
Briefly, PCR was performed with the mutation-specific primers under the following conditions: 35 cycles of 98 °C for 10 sec, 58 °C for 30 sec, and 72 °C for 5 min. After the PCR reaction, 5μl of the PCR products was digested with DpnI at 37 °C for 5 hours to remove the template plasmid, and then deactivated at 80 °C for 15 minutes. Next, recombination of the PCR products was induced with infusion recombinase.
After recombination, plasmids were transformed into Top10 competent cells (Thermo Fisher, MA, USA) for amplification. Sanger sequencing was used to confirm all variants.
Briefly, PCR was performed with the mutation-specific primers under the following conditions: 35 cycles of 98 °C for 10 sec, 58 °C for 30 sec, and 72 °C for 5 min. After the PCR reaction, 5μl of the PCR products was digested with DpnI at 37 °C for 5 hours to remove the template plasmid, and then deactivated at 80 °C for 15 minutes. Next, recombination of the PCR products was induced with infusion recombinase.
After recombination, plasmids were transformed into Top10 competent cells (Thermo Fisher, MA, USA) for amplification. Sanger sequencing was used to confirm all variants.
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