BLESS was performed as described in Crosetto et al., 2013 (link). Briefly, cells were fixed with 2% formaldehyde to stabilize chromatin and prevent artificial DSBs, plasma membranes were lysed, and intact nuclei were recovered by centrifugation. Nuclei were deproteinated using Proteinase K. DSBs were then blunted and 5′phosphorylated using the Quick Blunting Kit (NEB) and ligated to a biotinylated linker (proximal) using T4 ligase (NEB). Total DNA was extracted by precipitation with isopropanol and fragmented by sonication using a Covaris S220 to create ∼400 bp fragments. Labeled fragments were captured by streptavidin beads (Invitrogen) and once again blunted and phosphorylated using the Quick Blunting Kit (NEB), and ligated to a second linker (distal). The resulting circular DNA was linearized by I-SceI (NEB) digestion and amplified by PCR. Sequencing libraries were prepared using TruSeq Nano DNA LT Library Preparation Kit (Illumina). Quality and quantity of libraries were assessed on 2100 Bioanalyzer (Agilent) using High Sensitivity DNA Kit (Agilent), and on Qubit 2.0 using Qubit dsDNA HS Assay Kit (ThermoFisher).
Quick blunting kit
Manufactured by New England Biolabs
Sourced in United States, Germany
The Quick Blunting Kit is a laboratory tool designed to efficiently blunt the ends of DNA fragments. It is used to prepare linear DNA for ligation or other downstream applications where blunt-ended DNA is required.
Automatically generated - may contain errors
Lab products found in correlation
T4 ligase, by New England Biolabs (4 mentions)
Quick ligation kit, by New England Biolabs (4 mentions)
Streptavidin beads, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (3 mentions)
Messageampii kit, by Thermo Fisher Scientific (2 mentions)
Truseq small rna library prep kit, by Illumina (2 mentions)
Biotinylated proximal linker, by Merck Group (2 mentions)
Distal linker, by Merck Group (2 mentions)
Truseq dna lt sample prep kit, by Illumina (2 mentions)
I scei, by New England Biolabs (2 mentions)
S220 ultrasonicator, by Covaris (2 mentions)
Nanodrop nd 8000, by Thermo Fisher Scientific (2 mentions)
Minelute kit, by Qiagen (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
T4 dna ligase enzyme, by New England Biolabs (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rsii platform, by Pacific Biosciences (1 mentions)
Dna template prep kit 3, by Pacific Biosciences (1 mentions)
Gaiix platform, by Illumina (1 mentions)
25 protocols using quick blunting kit
1
BLESS: Genome-Wide Mapping of DNA Double-Strand Breaks
2
Lentiviral Integration Analysis by qPCR
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LM-PCR experiments were performed according to a protocol previously reported [41 (link)] with some modifications. In brief, after 2 days of transfection, cells were directly lysed in HMW buffer (10 mM Tris–HCl [pH 7.5], 100 mM NaCl, 1 mM EDTA, 0.5% SDS) supplemented with ProteinaseK (200 μg/ml), and incubated overnight at 55 °C without agitation. After phenol–chloroform extraction, same volume of isopropanol was added to sample, and DNA was gently picked up and rinsed with 70% ethanol. Recovered DNA was suspended in 1×TE supplemented with RNaseA (100 μg/ml). Equal amounts of DNA was treated with Quick-Blunting Kit (NEB) and ligated with blunt-end linkers (JW-Linker: annealed JW102 with JW103). Following purification, equal amounts of DNA were subjected to qPCR analysis with LacO-Rev and JW102 primers using SyBr Premix ExTaq Tli RNaseH plus. For estimating the amounts of LacO/JW-linker junction, corresponding DNA fragment was cloned into pMD20, and used as standard sample. The qPCR against to β-globin was carried out in parallel to normalize the amounts of input DNA. Oligonucleotides used in this procedure are listed in (Additional file 20 : Table S3).
3
Multi-Sequencing Platform Genome Characterization
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DNA was extracted using the phenol chloroform protocol (Neumann et al., 1992 (link)). The genome was sequenced separately in three sequencing runs: (1) Using an Illumina GAIIx platform (72 bp paired-end read). The libraries for this run were produced as follows: DNA was sheared by sonication using a Bio-Ruptor ultrasonicator (Diagenode), end-repaired using the Quick Blunting kit from New England Biolabs, and Illumina adaptors were ligated using the quick ligate kit from Enzymatics followed by PCR enrichment. Size selection was performed using SPRI beads to obtain fragments with a mean size of 228 bp; (2) Using an Illumina HiSeq2000 platform (150 bp paired-end read). The libraries for this sequencing run were produced at the University of Haifa Bioinformatics Support Unit using the NEBNext Ultra DNA Library Prep Kit for Illumina followed by size selection using SPRI beads to obtain fragments with a mean size of 475 bp; (3) Using a Pacific Biosciences RS II platform, with the libraries produced by the DNA template prep kit 3.0 from Pacific Biosciences, at the Yale Center for Genome Analysis.
4
ATAD3A Gene Knockout via CRISPR-Cas9
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Single guide RNAs targeting ATAD3A (oligonucleotides used given in Table 1 ) were designed using the CRISPOR tool (crispor.tefor.net) and cloned into lentiviral construct lentiCRISPRv2-hygro, a gift from Brett Stringer (Flinders Health and Medical Research Institute, Adelaide, Australia; Stringer et al., 2019 (link); plasmid #98291; Addgene), following the protocol provided on the plasmid website. EVs 3 and 12 are two clones obtained after BSMBI digestion to remove buffer sequence, blunting and ligation using the Quick blunting kit (New England Biolabs), following the manufacturer’s instructions. Lentiviral particles were produced, and THP-1 cells transduced as described for shRNA. 2 d after transduction, transduced cells were selected with 500 µg/ml hygromycin B (Invivogen), and selected cell pools were analyzed 7 d after transduction.
5
Transposon-Chromosome Junction Sequencing
6
BLISS Sequencing of ASiSI-ER-U20S Cells
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BLISS was performed on DIvA (AsiSI-ER-U20S) cells mock treated or induced with 4-OHT using one coverslip per condition (11 millimiters), containing ∼30.000 adherent cells. Briefly, 4 h after induction, cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. After washing, cells were lysed and submitted to in situ DNA ends blunting (with Quick Blunting kit, NEB), followed by in situ DNA ends ligation with BLISS linker (Supplementary Table 3 ). After washing, genomic DNA was extracted and sonicated with Covaris S220 (10% duty factor, 175 W peak incident power, 200 Cycles/burst, 105s) to obtain a pool of 300 bp fragments. Afterwards, fragmented DNA was in vitro transcribed using MessageAmpII kit (Ambion) for 14 h at 37 °C. After RNA purification and ligation of the 3′-Illumina adapter, the RNA was reverse transcribed. The final step of library indexing and amplification was performed using the Illumina TruSeq Small RNA Library Prep Kit.
7
Mapping Double-Strand DNA Breaks
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After 4OHT and or auxin treatment in G1 synchronized cells, DSBs labelling was carried out using BLESS method as described previously37 (link). Briefly, cells were fixed with formaldehyde, lysed and mildly digested with proteinase K at 37ºC in order to purify intact nuclei, then DSBs were blunted and 5'phosphorylated using Quick Blunting Kit (NEB). Subsequently, a biotinylated proximal linker (Sigma) was ligated to DSBs using T4 ligase (NEB). Next, DNA was extracted by precipitation with isopropanol and sonicated using Covaris S220 ultrasonicator to create fragments approximately 400bp long. Labeled, biotinylated fragments were captured on streptavidin beads (Invitrogen) and ligated to a distal linker (Sigma). Resulting DNA fragments were then linearized by I-SceI (NEB) digestion and amplified by PCR. Libraries were prepared using TruSeq DNA LT Sample Prep Kit (Illumina), followed by 2x61 bp and 2x70 bp next generation sequencing on Illumina HiSeq 2500. Raw data were filtered using BLESS adapter in order to keep only the end proximal to the cut from the paired read. These proximal reads were aligned using bwa-aln and PCR-duplicates were cleaned using samtools. The coverage and the normalization by the total number of reads was then generated using R. For each site we computed the average level of normalized counts in a window of +/-500bp around sites.
8
In Situ DNA Mapping and Transcription
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BLISS was performed as described in detail in Yan et al. (2017) (link) (Mirzazadeh et al., 2018 (link)). Briefly, :300,000 cells were grown on 12mm coverslips in 24-well plates. After treatment (or mock), cells were washed twice with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. After washing, cells were lysed and submitted to in situ DNA ends blunting (with Quick Blunting kit, NEB), followed by in situ DNA ends ligation with BLISS linker. After washing, genomic DNA was digested by HaeIII (NEB) for 3hr at 37°C and the fragmented DNA was extracted. The fragmented DNA was in vitro transcribed using MessageAmpII kit (Ambion) for 14 h at 37°C. After RNA purification and ligation of the 3′-Illumina adaptor, the RNA was reverse transcribed. The final step of library indexing and amplification was performed using the Illumina TruSeq Small RNA Library Prep Kit.
9
Cloning Unstable DNA Fragments
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Purified DNA products were cloned into pJAZZ-OCmin, a derivative of the linear pJAZZ-OC vector (Lucigen, Middleton, WI), which is optimal for cloning unstable DNA fragments (Fig 4 ; [21 (link)]). pJAZZ-OCmin lacks several genes of the parental vector, most notably telN, which is responsible for maintenance of the linear structure [22 (link)]. TelN activity is provided in trans by the E. coli host strain TSA G5, which is a TelN-overexpressing derivative of the BigEasy TSA strain (Lucigen). The remaining N15 genes that were deleted in the construction of pJAZZ-OCmin are not essential for plasmid function [21 (link), 22 (link)]. To prepare the PCR product for cloning, it was blunt ended using the Quick Blunting Kit (New England Biolabs, Ipswich, MA) and ligated to the left and right arms of the plasmid backbone, with subsequent transformation into TSA G5 electrocompetent cells. Transformed clones were selected with 12.5 microgram/ml chloramphenicol and grown at 16°C to maintain stability of the expanded repeat. Several independent transformants were chosen for further analysis of insert size via gel electrophoresis of the cloned insert. The clone containing the longest repeat expansion was selected for SMRT sequencing.
10
Mapping Double-Strand DNA Breaks
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