The MPRA oligo library was amplified using Q5 High-Fidelity DNA Polymerase (NEB). The amplified library was size-selected using Agencourt AMPureXP beads (Beckman Coulter). The oligo library was then inserted into an empty MPRA vector by golden gate assembly using BsaI (NEB) and T4 Ligase (NEB). The resulting library was purified using isopropanol precipitation (15 µL elute) then expanded by electroporation into 5 vials (3 µL ligation/vial) of One Shot TOP10 Electrocomp E. coli (ThermoFisher). The plasmid library was isolated using the Plasmid Plus Maxi kit (QIAGEN). The MPRA reporter gene (SV40 promoter followed by GFP) was then incorporated into the plasmid library by golden gate assembly using Esp3I (NEB) and T4 Ligase (NEB). The final MPRA plasmid library was purified, expanded, and isolated as described previously. Primers and PCR conditions are listed in Supplemental Table S6 .
T4 ligase
Manufactured by New England Biolabs
Sourced in United States, Germany, United Kingdom, China, Israel
T4 ligase is an enzyme that catalyzes the formation of phosphodiester bonds between adjacent 3' hydroxyl and 5' phosphate termini in DNA or RNA. It is commonly used in molecular biology protocols such as DNA cloning and library construction.
Automatically generated - may contain errors
Lab products found in correlation
Restriction enzyme, by New England Biolabs (30 mentions)
T4 polynucleotide kinase, by New England Biolabs (24 mentions)
Qiaquick gel extraction kit, by Qiagen (23 mentions)
Bamhi, by New England Biolabs (17 mentions)
Ecori, by New England Biolabs (16 mentions)
T4 ligase buffer, by New England Biolabs (16 mentions)
Q5 polymerase, by New England Biolabs (16 mentions)
T4 pnk, by New England Biolabs (15 mentions)
Restriction endonuclease, by New England Biolabs (15 mentions)
Qiaprep spin miniprep kit, by Qiagen (11 mentions)
Phusion polymerase, by New England Biolabs (11 mentions)
Xhoi, by New England Biolabs (10 mentions)
Qiaquick pcr purification kit, by Qiagen (10 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (9 mentions)
Dpni, by New England Biolabs (9 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (8 mentions)
Hiseq 2500, by Illumina (7 mentions)
Ampure xp bead, by Beckman Coulter (7 mentions)
Dna polymerase, by New England Biolabs (7 mentions)
Antarctic phosphatase, by New England Biolabs (6 mentions)
519 protocols using t4 ligase
1
MPRA Library Construction Protocol
2
High-throughput 4C Chromatin Capture
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The 4C experiments were carried out using a published protocol51 (link) with some modifications. For each assay, 1 × 107 cells were cross-linked and quenched as in ChIP assays. Nuclei were isolated in a 50 ml of lysis buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.2% IGEPAL CA-630 detergent and 1 × protease inhibitors). Subsequently, the nuclei were digested with 240 U NlaIII enzyme (New England BioLabs Inc., R0215L) followed by an overnight in-nuclei ligation with 4,000 U T4 ligase (New England BioLabs Inc., M0202M) at 16 °C. The ligated DNA was de-crosslinked, purified, digested with 90 U CviQI enzyme (New England BioLabs Inc., R0639S) and circularized by 5,000 U T4 ligase. The circularized DNA (16 × 300 ng) was amplified with bait-specific inverse primers (Supplementary Table 3 ), pooled and purified, followed by KAPA Hyper Prep protocols.
3
Overexpression and Silencing Plasmid Construction
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We constructed overexpression vectors for DNAAF5, PFKL and USP39 genes using pCDNA3.1-puro-Flag, pCDNA3.1-G418-myc and pCDNA3.1-Hygro-HA plasmids, respectively. The extracted RNA was reverse transcribed to cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd), after which the corresponding fragment was amplified using 2 × Phanta Flash Master Mix (Dye Plus) (Vazyme Biotech Co., Ltd) and a ClonExpress II One The Step Cloning Kit (Vazyme Biotech Co., Ltd) to ligate the fragment to the corresponding vector. Complete plasmids were used for subsequent experiments after sequences had been verified to be correct.
The LentiCRISPRv2 plasmid was digested using Esp3I (R0734, NEB), after which the annealed primer fragment was ligated into it using T4 ligase (M0202, NEB).
The PLKO.1-Hygro vector was used to silence the corresponding gene. After double-digestion with AgeI-HF (R3552, NEB) and EcoRI-HF (R3101, NEB), T4 ligase (M0202, NEB) was used to link the annealed primer fragments into the vector.
Depending on the progress and needs of the experiment, the Lipo8000 (C0533, Beyotime) transfection reagent was used to transfer different plasmids into various cells after which corresponding antibiotics were added to screen and obtain equivalent stable cell lines.
The LentiCRISPRv2 plasmid was digested using Esp3I (R0734, NEB), after which the annealed primer fragment was ligated into it using T4 ligase (M0202, NEB).
The PLKO.1-Hygro vector was used to silence the corresponding gene. After double-digestion with AgeI-HF (R3552, NEB) and EcoRI-HF (R3101, NEB), T4 ligase (M0202, NEB) was used to link the annealed primer fragments into the vector.
Depending on the progress and needs of the experiment, the Lipo8000 (C0533, Beyotime) transfection reagent was used to transfer different plasmids into various cells after which corresponding antibiotics were added to screen and obtain equivalent stable cell lines.
4
CRISPR-Cas9 Vector Construction for Ara h 2 Gene
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The 35S:GFP and CmYLCV:GFP vectors were used for checking the transformation efficiency in this study. Three intermediate module plasmids A, B, and C were prepared for the construction of the CRISPR–Cas9 vector of Ara h 2 [46 (link)]. For module A, CmYLCV promoter from pMOD_A3003 (Addgene #91043) was inserted into pMOD_A0101 (Addgene #90998) in place of 35S promoter via restriction digestion and cloned using T4 Ligase (NEB, Ipswich, MA, USA) (Figure S2A,B ). The pMOD_B2303 vector was used for module B. The polycistronic tRNA–gRNA (PTG) gene containing two sgRNAs sequences for Ara h 2 [47 (link)] was synthesized and incorporated commercially into pUC57 (Genscript Biotech Ltd., Piscataway, NJ, USA). The synthesized pUC57-PTG was digested with PstI and XhoI and cloned into the PstI and XhoI-digested pMOD_B2303 vector (Addgene #91068) using T4 Ligase (NEB) following the manufacturer’s recommendations (Figure S2A,C ). Modified pMOD_A0101, modified pMOD_B2303, and empty vector pMOD_C0000 (Addgene #91081) were assembled into a non-binary vector, pTRANS_100 (Addgene #91198) by simple Golden Gate protocol using the AarI enzyme [47 (link)] (Figure S2A,D ).
5
Construction of guide+donor library
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The CustomArray-synthesized oligo library was diluted to 1 ng/μl and 1μl of the library was amplified with Kapa SYBR FAST qPCR Kit Master Mix (Kapa Biosystems) using unique primer pairs specific to each desired library (e.g. SGS1 tiling deletion, smORF library, etc.). Primers used for oligo library amplification were further modified to contain the necessary overlaps to enable the library to be inserted into our vector backbone via Golden Gate cloning. The PCR products were run on a gel to confirm amplicons are of the expected length. After PCR purification (Zymo Research), the amplicon is cloned into the BsmBI-containing library vector (XG128) using a standard Golden Gate protocol with BsmBI (NEB R0580S) and T4 ligase (NEB M0202S) then electroporated into 5-alpha Electrocompetent E.coli cells (NEB C2989). This ensuing library now contained the guide and donor sequences adjacent to the SNR52 promoter but was still missing the sgtail and an RNA polIII terminator. To clone in the additional functional components between the guide and donor, we amplified and cloned in the sgtail and terminator sequences following the same Golden Gate cloning method as described above, but this time using SapI (NEB R0569S) and T4 ligase. The resulting Golden Gate reactions were then PCR purified and electroporated into 5-alpha Electrocompetent E.coli cells to create a final guide+donor library.
6
Constructing Retroviral Vectors for Gene Expression
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Addgene plasmid #102919 (https://www.addgene.org/102919/ ) was used to express ABE7.10 for the in vitro experiments. For U6-sgRNA expression plasmids, five different gRNA oligonucleotides were synthesised (Sigma) and cloned into the pU6gRNA vector using the BsmBI restriction site (Supplementary Table 2 ). To generate the retroviral construct for the GFP switch-on system, gfp was PCR-amplified from pSERS11 SF GFP24 (link) and modified by using a long primer containing Gly-Ser stretch, the stop codon TAG and the restriction site for NcoI (Sigma; P1 and P2 in Supplementary Table 1 ). The backbone and the PCR product were digested using NcoI and NotI (NEB) and ligated with T4 ligase (NEB).
Similarly, to generate the retroviral construct for the HFE-GFP switch-on system, gfp was PCR-amplified from pSERS11 SF GFP using a long primer containing 20 bp of the HFE sequence, carrying the stop codon TAG, a Gly-Ser stretch and the restriction site for NcoI (Sigma; P3 and P2 in Supplementary Table1 ). The backbone and the PCR product were digested using NcoI and NotI (NEB) and ligated with T4 ligase (NEB). Amplification of plasmids was performed in One Shot™ Stbl3™ Chemically Competent E. coli (C737303, Invitrogen).
Similarly, to generate the retroviral construct for the HFE-GFP switch-on system, gfp was PCR-amplified from pSERS11 SF GFP using a long primer containing 20 bp of the HFE sequence, carrying the stop codon TAG, a Gly-Ser stretch and the restriction site for NcoI (Sigma; P3 and P2 in Supplementary Table
7
Constructing pIL-SVnisBTC and Derivatives
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The plasmid pIL-SVnisBTC was generated from pIL-SV and pIL3BTC31 (link). The plasmid pIL3BTC was digested with the restriction enzymes NotI (NEB) and BstXI (NEB) to receive a fragment BTC containing the genes nisB, nisT and nisC. Next, pIL-SV62 (link) was also digested with NotI and BstXI (pIL-SV**). The fragment BTC and pIL-SV** were ligated with T4-ligase (NEB) and transformed into E. coli DH5α. The sequence of the construct pIL-SVnisBTC (Table S5 ) was verified by DNA sequencing (Microsynth Seqlab).
By using Phusion DNA polymerase (NEB) with the appropriate primer pairs (TableS4 ) the gene deletions of nisB, nisC or nisT were performed to generate pIL-SVnisBTC derivatives. Subsequently, the linearized vectors were ligated with T4-ligase (NEB) and transformed into E. coli DH5α. The sequence of the constructs (Table S5 ) were verified by DNA sequencing (Microsynth Seqlab).
To generate nisTH551A and nisCH331A mutants, a polymerase chain reaction using PfuUltra II Fusion DNA polymerase (Agilent Technologies), the template pIL-SVnisBTC and the appropriate pair of oligonucleotides (TableS4 ) was performed according to standard procedures. The sequence of the new constructs (Table S5 ) were verified by DNA sequencing (Microsynth Seqlab).
By using Phusion DNA polymerase (NEB) with the appropriate primer pairs (Table
To generate nisTH551A and nisCH331A mutants, a polymerase chain reaction using PfuUltra II Fusion DNA polymerase (Agilent Technologies), the template pIL-SVnisBTC and the appropriate pair of oligonucleotides (Table
8
Construction of guide+donor library
9
Chromatin Conformation Capture by 4C-seq
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In total, 1 × 107 A549 cells were used per 4C assay. Cells are treated with formaldehyde, which cross-links proteins to proteins and DNA. Cross-linked chromatin is subsequently digested with HindIII (NEB). Next, chromatin is diluted and then religated by T4 ligase (NEB) to fuse the ends of DNA fragments. DNA is further digested by DpnII (NEB) that digests the fragment into smaller fragments after the removal of cross-links by heating. These fragments are religated under diluted conditions to create much smaller DNA circles using T4 ligase (NEB) again. Inverse PCR primers with Illumina forward and reverse adaptors were designed to anneal to a bait locus HindIII/DpnII restriction fragment. A total of 3200 ng of 4C template was used to amplify each bait using Expand Long Template Polymerase (Roche) system. The PCR program is as follows: 2 min at 94 °C; 10 s at 94 °C; 1 min at bait specific annealing temperature; 3 min at 68 °C; 29 × repeat; 5 min at 68 °C; hold at 4 °C. All 16 PCR tubes were pooled and purified using High Pure PCR Product Purification Kit (Roche) kit. Subsequently, high-throughput sequencing is used to detect the captured regions.
10
Plasmid Generation through Restriction Digestion
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To generate plasmids AGB245/246, pAKH37 and AGB21 were digested with KpnI and NdeI (NEB). The ∼1600–base pair band (pAKH37) and the 4214–base pair band (AGB21) were gel extracted and ligated using T4 Ligase (NEB). Resulting plasmids were verified by digesting with KpnI and NdeI and sequencing. To generate plasmids AGB264/265, AGB246 and pRS416 were digested with NdeI and SbfI. The 3343–base pair band from AGB246 was gel extracted and ligated with digested pRS416 using T4 Ligase (NEB). Resulting plasmids were verified with SacI digests and sequencing with AGO37.
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