A total of 106 crosslinked nuclei were washed twice with Csp6I RE buffer (Thermo Scientific, Waltham, MA, USA, #ER0211) and resuspended in Csp6I RE buffer. SDS was added for a final concentration of 0.3% (v/v). After 30 min of incubation at 65 °C, Triton X-100 was added for a final concentration of 1.8% and the sample was incubated for 1 h at 37 °C. Then, 150 units of restriction enzyme Csp6I (Thermo Scientific, Waltham, MA, USA, #ER0211) were added and the sample was incubated overnight at 37 °C. The inactivation of Csp6I was achieved by incubating the samples at 65 °C for 25 min. Ligation reaction was then done in 1.5 mL of 1X ligase buffer with 25 units of T4 ligase (Roche, Rotkreuz, Switzerland, #EL0011) and incubated overnight at 4 °C, followed by 30 min of incubation at RT. To reverse the crosslinks, the sample was supplemented with 50 µg of proteinase K (Invitrogen, Waltham, MA, USA, #25530049) and incubated overnight at 65 °C, followed by incubation with 150 µg of RNAse (Thermo Scientific, Waltham, MA, USA, #EN0551) for 45 min at 37 °C. The resulting proximity-ligated DNA was extracted by phenol–chloroform followed by ethanol precipitation and dissolved in 20 µl of TE buffer. To generate DNA circles, DNA was then digested overnight with DpnII (NEB, Ipswich, MA, USA, #R0543T) at 37 °C.
Csp6i
Manufactured by Thermo Fisher Scientific
Sourced in Lithuania, United States
Csp6I is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GTAC-3'. It is commonly used in molecular biology applications such as DNA fragmentation, cloning, and restriction mapping.
Automatically generated - may contain errors
Lab products found in correlation
Dpnii, by New England Biolabs (10 mentions)
T4 dna ligase, by Thermo Fisher Scientific (6 mentions)
T4 dna ligase, by Roche (4 mentions)
Bsu15i, by Thermo Fisher Scientific (4 mentions)
Ecorii, by Thermo Fisher Scientific (3 mentions)
Ecorv, by Thermo Fisher Scientific (3 mentions)
Nextseq 500, by Illumina (3 mentions)
Ndei restriction endonucleases, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Nlaiii, by New England Biolabs (1 mentions)
Ultra 2 q5 master mix, by New England Biolabs (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Dpnii, by Thermo Fisher Scientific (1 mentions)
T4 ligase, by Roche (1 mentions)
Proteinase k, by Thermo Fisher Scientific (1 mentions)
Eco32i, by Thermo Fisher Scientific (1 mentions)
Bamhi, by Thermo Fisher Scientific (1 mentions)
Hindiii enzyme, by New England Biolabs (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
22 protocols using csp6i
1
Chromosome Conformation Capture Assay
2
Chromosome Conformation Capture Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4C sample preparation was performed as previously described by Splinter et al. using NlaIII (NEB) and Csp6I (Thermo)34 (link). Viewpoint primers were selected from a previously reported database35 (link).
Primers sequences were concatenated with Illumina compatible adapter sequences and unique barcodes and synthesized as Ultramer oligos (IDT). Final library amplification was set up on ice using 3.2 µg of purified 4C sample DNA, 2.5 µM primers, and Ultra II Q5 Master Mix (NEB) split into 16 × 50 µl PCR reactions with extension at 65 °C. Amplification products were purified using 1.5× Ampure XP (Beckman) bead purification. Size distributions were established using a Bioanalyzer DNA 12000 Assay (Agilent).
Paired-end sequencing reads were generated as above and analyzed with the 4C-ker pipeline as previously described to identify genomic loci with differential chromosome contacts between conditions36 (link).
Primers sequences were concatenated with Illumina compatible adapter sequences and unique barcodes and synthesized as Ultramer oligos (IDT). Final library amplification was set up on ice using 3.2 µg of purified 4C sample DNA, 2.5 µM primers, and Ultra II Q5 Master Mix (NEB) split into 16 × 50 µl PCR reactions with extension at 65 °C. Amplification products were purified using 1.5× Ampure XP (Beckman) bead purification. Size distributions were established using a Bioanalyzer DNA 12000 Assay (Agilent).
Paired-end sequencing reads were generated as above and analyzed with the 4C-ker pipeline as previously described to identify genomic loci with differential chromosome contacts between conditions36 (link).
3
4C-seq Protocol for BLV Integration Sites
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4C-seq experiments were performed as previously described (52 (link)). Briefly, 107 cells per sample were cross-linked in 2% formaldehyde for 10 min and the reaction was quenched by adding glycine at a final concentration of 0.13 M. Then, cells were lysed and the harvested cross-linked chromatin was digested using the restriction enzyme MboI (NEB). After a ligation step with the T4 DNA ligase (NEB), ligated samples were decross-linked and subjected to a second round of digestion using the restriction enzyme Csp6I (Thermo Scientific). Digested samples were then ligated using T4 DNA ligase (Roche) and purified before being used as a template for inverse PCR. Primers used for inverse PCR and library preparation are listed in Supplementary Table S2 . Products were sequenced using Illumina sequencing (Illumina NextSeq 500) and reads were mapped to a hybrid ovine genome (OAR v3.1) containing the BLV provirus sequence (GenBank: KT122858.1 ) at the L267 or YR2 integration sites (Supplementary Table S4 ) and processed using pipe4C (52 (link)) (github.com/deLaatLab/pipe4C) with the following parameters: normalization to 1 million reads in cis, window size 21, top 2 read counts removed. Peaks were called using peakC (53 (link)) using default settings (alphaFDR = 0.1, qWr = 1). Coverage plots were generated using R (https://www.R-project.org/ ).
4
4C Sequencing from A549 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4C template preparation from 5 million A549 cells treated as indicated in the figure legend was done as described in reference 47 (link) using Csp6I (Thermo Fisher Scientific) and DpnII (Thermo Fisher Scientific) as primary and secondary restriction enzymes, respectively. Sequencing library preparation was essentially performed as described in reference 68 (link), with the exception that a 1.5× AMPure XP purification was carried out after the first PCR. Primer pairs used for the inverse PCR are listed in Table 3 . 4C libraries were submitted for single-end Illumina sequencing.
5
Phage DNA Extraction and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The aliquot of high-titer (1011–1012 PFU/mL) phage suspension was subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [33 ]. The isolated phage DNA was subsequently used for PCR and restriction analysis, or it was subjected to genome sequencing. The restriction digestion was performed with Bsu15I, Csp6I, DraI, EcoRII, EcoRV, HhaI, MboI and NdeI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. A restriction analysis was performed in triplicate to confirm the results.
6
Genome-wide Chromatin Interaction Mapping
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4C was performed as described (24 (link),25 (link)). Proximity ligation junctions, reflecting in vivo spatial proximity, were generated with DpnII (New England Biolabs), followed by circularization with Csp6I (Thermo Scientific). Chromosomal contacts with the TSS-containing fragment were amplified with inverse PCR primers (Supplementary Table S1 ), and sequenced on the Illumina 2000 platform.
7
4C-seq on Embryonic Stem Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4C-seq was performed on 5 million to 10 million feeder-fee EpiSCs or ESCs according to published protocols (Splinter et al. 2012 (link)). A first digest was performed with DpnII (New England Biolabs); a second digest was performed with Csp6I (ThermoFisher). PCR primers were designed using guidelines described previously (Splinter et al. 2012 (link)) and are listed in Supplemental Table S4 . Samples were run by the USF on an Illumina NextSeq500. After mapping on a reduced mm9 genome (van de Werken et al. 2012 (link)), the highest covered fragment was removed, and the data set was normalized to 1 million intrachromosomal reads.
8
Phage DNA Extraction and Restriction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The aliquots of high-titer (1011–1012 PFU/mL) phage suspension were subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [84 ]. The isolated phage DNA was subsequently used in restriction analysis, for PCR, or it was subjected to genome sequencing. The restriction digestion was performed with Bsu15I, Csp6I, DraI, EcoRII, EcoRV, HhaI, MboI, and NdeI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. The restriction analysis was performed in triplicate and Figure 4 shows a representative result.
9
Phage DNA Extraction and Restriction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The aliquots of phage suspension (1011–1012 PFU/mL) were subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [22 ]. The isolated phage DNA was subsequently used in restriction analysis, for PCR, or it was subjected to genome sequencing. The restriction digestion was performed with BamHI, Bsu15I, Csp6I, DraI, EcoRII, Eco32I, HhaI, MboI, MfeI, NdeI, and RsaI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. A restriction analysis was performed in triplicate to confirm the results.
10
Chromatin Conformation Capture (4C) Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
4 C was performed as previously described58 (link),59 (link). Cells were fixed with 2% formaldehyde for 10 min, cross-linked chromatin was digested overnight with an excess of HindIII enzyme (New England Biolabs) and then DNA ends were ligated under dilute conditions that favor junctions between cross-linked DNA fragments. The ligation junctions were then circularized by digestion with Csp6I (Thermo Scientific). Chromosomal contacts with the baits were amplified with inverse PCR primers (Supplementary Table S2 ) using Platinum Taq DNA Polymerase (LifeTechnologies). 4C-seq libraries were sequenced on Illumina Hi-seq 2000 platform.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!