Genomic DNA was extracted from wheat leaves using Plant DNAzol Reagent (Invitrogen) and then fragmented. The DNA ends were repaired using the End-It kit (Lucigen) and then an A-tail was added using the Klenow fragment (3′–5′ exo-; NEB). The truncated Illumina Y-adapter (Annealed by using adaptor strand A: 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ and adaptor strand B: 5’-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’, where ‘P’ indicates a 5’ phosphate group) was ligated to the DNA using T4 DNA ligase (Promega). Full-length TF coding sequences were cloned into the pIX-Halo vector. For TFs with multiple isoforms, the longest coding sequence was selected. Halo-tagged TFs were expressed in vitro using the TNT SP6 Coupled Wheat Germ Extract System (Promega) and then immobilized using Magne HaloTag Beads (Promega) before they were incubated with the DNA library. The DNA binding to specific TFs was eluted for 10 min at 98 °C and then amplified by PCR using indexed Illumina primers and Phanta Max Super-Fidelity DNA Polymerase (Vazyme). To capture the background DNA, the Halo tag encoded in the empty pIX-Halo vector (i.e., without a TF coding sequence) was expressed and incubated with the DNA library. The amplified fragments were purified using VAHTS DNA Clean Beads (Vazyme) and then sequenced by Novogene (Beijing, China) using the Illumina NovaSeq 6000 system to produce 150-bp paired-end reads.
Tnt sp6 coupled wheat germ extract system
Manufactured by Promega
Sourced in United States
The TNT SP6 Coupled Wheat Germ Extract System is a laboratory product that enables in vitro transcription and translation of recombinant proteins. It provides a cell-free system derived from wheat germ that can be used to produce proteins from DNA templates.
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Lab products found in correlation
Magne halotag beads, by Promega (8 mentions)
Novaseq 6000 system, by Illumina (2 mentions)
Vahts dna clean beads, by Vazyme (2 mentions)
Phanta max super fidelity dna polymerase, by Vazyme (2 mentions)
T4 dna ligase, by Promega (2 mentions)
Pdonr221 vector, by Thermo Fisher Scientific (2 mentions)
Lr clonase 2, by Thermo Fisher Scientific (2 mentions)
M220 ultrasonicator, by Covaris (2 mentions)
Navoseq, by Illumina (2 mentions)
Plant dnazol reagent, by Thermo Fisher Scientific (1 mentions)
Dna retardation gel, by Thermo Fisher Scientific (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Odyssey emsa buffer kit, by LI COR (1 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris protein gel, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 affinity gel, by Merck Group (1 mentions)
Nextflex adaptors, by PSC Biotech (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Kapa real time library amplification kit, by Roche (1 mentions)
16 protocols using tnt sp6 coupled wheat germ extract system
1
Wheat Leaf DNA Extraction and TF Binding
2
In Vitro Protein Expression
3
Quantifying MYC and MAX Protein Interactions
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MYC and MAX were translated individually or together in vitro using the TnT SP6 coupled wheat germ extract system (Promega), according to the manufacturer’s protocol. Plasmids used for MYC and MAX were pCS2-FLAG-hMYC and pRK7-HA-hMAX, respectively, and were generously provided by the Eisenman Lab (Fred Hutchinson Cancer Research Center). The protein concentrations of the in vitro translated products were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Binding reactions were carried out using Odyssey EMSA Buffer Kit (LI-COR), where 90–100 µg of the translated proteins were incubated with 7.5 nM IRDdye 700-labeled FGF7 WT or mutant DNA probes (IDT) in the presence or absence of their respective unlabeled competitor oligos (IDT), according to the manufacturer’s protocol. To separate the DNA–protein complex, the binding reactions were subjected to electrophoresis on a 6% DNA retardation gel (Thermo Fisher Scientific), which was then scanned using the Odyssey Infrared Imaging System (LI-COR) to detect the fluorescence signal. The assay was performed three times and showed similar results.
4
Affinity Purification of HA- and FLAG-Tagged Proteins
5
Halo-tagged MeGI and SiMeGI protein preparation
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The DAP genomic DNA libraries were prepared as previously described [25 (link), 28 (link), 35 (link)]. Briefly, the Covaris M220 ultrasonicator (with the manufacturer-recommended setting) was used to fragment gDNA to an average size of 200 bp. The resulting fragmented gDNA was ligated to the NEXTflex adaptors (Bioo Scientific, Austin, USA) as described, to make genomic libraries. The full-length SiMeGI cDNA was cloned into the pDONR221 vector (Life Technologies) and then transferred to the pIX-Halo using LR clonase II (Life Technologies) to generate pIX-Halo-SiMeGI. pIX-Halo-MeGI has been constructed previously [25 (link)]. The N-terminally Halo-tagged MeGI and SiMeGI were produced using the TNT SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA) and purified with Magne HaloTag beads (Promega). A total of 50 ng DAP gDNA library was incubated with Halo-tagged MeGI and SiMeGI at room temperature for 1 h.
6
DNA Affinity Purification Sequencing of FmoAFT
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DAP‐seq was conducted at Biorun Biotech, following the procedure described in a previous study (O'Malley et al., 2016 (link)). Genomic DNA (gDNA) was extracted from the leaves of female seedlings of F. moupinensis. The fragmented gDNA was used to construct libraries using the NEXTflex Rapid DNA‐Seq Kit (Bioo Scientific, USA). To clone the full‐length FmoAFT cDNA, the pFN19K HaloTag T7 SP6 Flexi (Promega, USA) vector was employed. The Halo‐FmoAFT fusion protein was subsequently expressed using the TNT SP6 Coupled Wheat Germ Extract System (Promega). Then the correctly expressed proteins were identified by Western blotting and purified using Magne Halo Tag Beads (Promega). The FmoAFT‐bound beads were incubated with adapter‐ligated gDNA libraries and amplified through PCR, utilizing the KAPA Real‐Time Library Amplification Kit (KAPA Biosystems, USA). The purified DNA fragments were sequenced on an Illumina NovaSeq 6000 platform (Illumina, USA). As a negative control, mock DAP‐seq libraries were prepared without the addition of proteins to the beads. DAP‐seq reads were aligned to the F. moupinensis genome using the mem method of BWA (Li and Durbin, 2010 (link)) with default parameters. DAP‐seq peaks were detected by MACS2 (Zhang et al., 2008 (link)). Conservative motifs within the peak regions were analysed using HOMER (Heinz et al., 2010 (link)) and GEM (Guo et al., 2012 (link)).
7
Genome-wide Identification of TF Binding Sites
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DAP-seq was performed as previously described (Bartlett et al. 2017 (link)). Genomic DNA was extracted from wheat leaves using Plant DNAzol Reagent) and fragmented. DNA was then end-repaired using the End-It kit (Lucigen) and A-tailed using Klenow (3′–5′ exo-; NEB). Truncated Illumina Y-adapter was ligated to DNA using T4 DNA Ligase (Promega). Full-length TF was cloned into pIX-Halo vector. Halo-tagged TF was expressed in vitro using the TNT SP6 Coupled Wheat Germ Extract System (Promega). Halo-TF was immobilized by Magne HaloTag Beads (Promega) and then incubated with the DNA library. TF-specific binding DNA was eluted for 10 min at 98°C and amplified with indexed Illumina primer using Phanta Max Super-Fidelity DNA Polymerase (Vazyme). Meanwhile, to capture background DNA which captured by Halo, pIX-Halo vector without TF cloned was expressed and incubated with the DNA library as well. The PCR product was purified using VAHTS DNA Clean Beads (Vazyme) and then sequenced by Novogene with the Illumina NovaSeq 6000 system to produce 150-bp paired-end reads.
8
Cloning and Expressing PeWRKY1 Protein
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The coding sequencing of PeWRKY1 was cloned into a pFN19K HaloTag T7 SP6 Flexi expression vector. Halo–PeWRKY1 fusion protein was expressed using the TNT SP6 Coupled Wheat Germ Extract System (Promega) following the manufacturer’s specifications for expression in a 50 μl reaction with a 2 h incubation at 37°C. Expressed proteins were directly captured using Magne Halo Tag Beads (Promega).
9
DAP-Seq Library Preparation and Sequencing
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The DAP genomic DNA library was prepared and the DAP reaction was completed as previously described (O'Malley et al., 2016 ; Bartlett et al., 2017 ). Briefly, the Covaris M220 ultrasonicator (with the manufacturer‐recommended setting) was used to fragment gDNA to an average size of 200 bp. The resulting fragmented gDNA was end‐repaired with the End‐It DNA Repair kit (Epicentre, Madison, WI, USA). Next, a dA‐tail was added using the Klenow fragment (3′→5′ exo‐) (NEB). The DAP‐Seq adaptor (i.e., truncated Illumina TruSeq adapter) was ligated to the fragmented gDNA with T4 DNA polymerase (NEB). Full‐length MeGI cDNA was cloned into the pDONR221 vector (Life Technologies) and then transferred to pIX‐Halo using LR clonase II (Life Technologies) to generate pIX‐Halo‐MeGi. The N‐terminally Halo‐tagged MeGI was produced using the TNT SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA) and purified with Magne Halo‐Tag beads (Promega). In total, 50 ng DAP gDNA library was incubated with Halo‐tagged MeGI at room temperature for 1 h. The recovered library was sequenced with the Illumina HiSeq 4000 system at the Vincent J. Coates Genomics Sequencing Laboratory at UC Berkeley.
10
Genomic DNA Binding Assay for OsMYB80 Transcription Factor
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The DAP-seq analysis was performed according to the protocol described by Bartlett et�al. (2017) (link). Briefly, 5 μg HHZ genomic DNA was sheared into ∼200-bp fragments and then ligated with the Illumina-based sequencing adaptors to form a DNA library. The library was examined for adaptor-ligation frequency by quantitative PCR (qPCR) before applied to DAP-seq assay.
OsMYB80 ORF fused to the Halo affinity tag was expressed in vitro using TNT� SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA). The HaloTag-OsMYB80 protein was then purified from nonspecific proteins in the expression system using the magnetic HaloTag ligand (chloroalkane) beads (Promega) and verified by Western blotting with the anti-HaloTag antibody (Promega). The purified protein was incubated with 500 ng adaptor-ligated genomic DNA library at 30�C for 2 h before washing away the unbound DNA fragments.
The samples were heated at 98�C for 10 min to release the OsMYB80-bound DNA, and the recovered DNA was then PCR-amplified with the indexed TruSeq primers (Illumina, San Diego, CA, USA). Indexed DNA samples were subsequently combined and size-selected to remove the residual adaptor dimers. Purified DNA libraries were then sequenced using the Illumina HiSeq sequencing platform.
OsMYB80 ORF fused to the Halo affinity tag was expressed in vitro using TNT� SP6 Coupled Wheat Germ Extract System (Promega, Fitchburg, WI, USA). The HaloTag-OsMYB80 protein was then purified from nonspecific proteins in the expression system using the magnetic HaloTag ligand (chloroalkane) beads (Promega) and verified by Western blotting with the anti-HaloTag antibody (Promega). The purified protein was incubated with 500 ng adaptor-ligated genomic DNA library at 30�C for 2 h before washing away the unbound DNA fragments.
The samples were heated at 98�C for 10 min to release the OsMYB80-bound DNA, and the recovered DNA was then PCR-amplified with the indexed TruSeq primers (Illumina, San Diego, CA, USA). Indexed DNA samples were subsequently combined and size-selected to remove the residual adaptor dimers. Purified DNA libraries were then sequenced using the Illumina HiSeq sequencing platform.
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