A modified version of the Illumina protocol was followed to prepare ChIP DNA samples for the deep sequencing pipeline. Briefly, blunting of the fragments was performed using the END-IT DNA repair kit (Epicentre) followed by the addition of a dA overhang using exo-minus Klenow (Epicentre). Paired-end adapters (Illumina) were ligated using the fast link kit (Epicentre). The fragments were amplified twice using the Illumina PE primers and PfuUltra II Fusion HS DNA polymerase (Stratagene), and each round of PCR was followed by gel purification and sizing of the fragments. Samples were cloned using the Topo cloning system (Invitrogen) and several clones were sequenced to assess sample quality prior to submission for sequencing on the Illumina GAII (Exp 1) or HiSeq 2000 (Exp 2) platforms at the UMASS Deep Sequencing Core facility, obtaining either 36 bp single-end (Exp 1) or 50 bp paired-end reads (Exp 2 and Pol2).
Topo cloning system
Manufactured by Thermo Fisher Scientific
The TOPO cloning system is a molecular biology tool used for the rapid and efficient insertion of DNA fragments into plasmid vectors. It utilizes the enzymatic activity of topoisomerase I to facilitate the direct ligation of PCR products into the vector, eliminating the need for traditional DNA ligase-based cloning methods. The TOPO cloning system provides a simple and streamlined approach for the cloning of various DNA sequences, enabling researchers to quickly generate recombinant plasmids for a wide range of applications.
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Lab products found in correlation
Peg it virus precipitation solution, by System Biosciences (2 mentions)
End it dna repair kit, by Illumina (1 mentions)
Paired end adapter, by Illumina (1 mentions)
Rneasy mini kit, by Bio-Rad (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Mutation surveyor software, by SoftGenetics (1 mentions)
X tremegene hp dna transfection reagent, by Roche (1 mentions)
Nepa21 electroporator, by Nepa Gene (1 mentions)
Pentr topo vector, by Thermo Fisher Scientific (1 mentions)
5 protocols using topo cloning system
1
ChIP-Seq Library Preparation Protocol
2
BCR-ABL1 Kinase Domain Sequencing
3
CEACAM5 and CEACAM6 Expression Analysis
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CEACAM5 and CEACAM6 cDNAs prepared from C45 CTOSs were amplified by PCR and the PCR products cloned into the TOPO cloning system (Invitrogen). After sequencing, the cDNAs were transferred to pCAGGS and 3x HA tag sequences inserted downstream of the signal sequence. Plasmid constructs were transfected into cells using X-tremeGENE HP DNA transfection reagent (Roche). For RNAi experiments, C45 CTOSs were incubated with 5 mM EDTA/PBS for 30 min at room temperature, followed by transduction of 150 pmole of Silencer select siRNAs (ABI, Waltham, MA) into 5000 CTOSs (40–70 μm) using a NEPA21 electroporator (Nepa Gene, Chiba, Japan). Three days after transduction of siRNAs, CTOSs were harvested and subjected to Western blotting.
4
Lentiviral Transduction of BMDMs
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BMDMs were transiently transfected using lentiviral technology (Invitrogen) as previously described [34] (link). Briefly, GFP-Rab5:S34N and GFP-2xFYVE were amplified from pEGFP-Rab5:S34N and pEGFP-2xFYVE vectors, respectively. Then, GFP, GFP-Rab5:S34N and GFP-2xFYVE were subcloned into the entry clone pENTR TOPO vector using the TOPO cloning system, as recommended by the manufacturer (Invitrogen). The inserted DNA was transferred into the destination vector to create a lentivirus encoding GFP, GFP-Rab5:S34N, GFP-2FYVE using the ViraPower HiPerform Gateway Expression System (Invitrogen), as recommended by the manufacturer. Viruses were produced using 293T cells, and viral supernatants were harvested 48 and 72 hours after transfection. The supernatants were centrifuged at 1,600×g for 15 minutes at 4°C, filtered through 0.45-µm filters, collected and concentrated using PEG-it virus precipitation solution (System Biosciences), and then stored at −80°C.
5
Mapping T-DNA Insertion Sites Using TAIL-PCR
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To identify the location of T-DNA insertions within the genomes of all mutants, thermal asymmetric interlaced PCR (TAIL-PCR) was performed on genomic DNA using the method and nonspecific primer sequences previously described by Liu et al. (1995) (link) and primers within the T-DNA. The resulting PCR products were cloned into the vector PCR4 using the TOPO cloning system (Invitrogen) and sequenced.
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