CIRCLE-seq libraries49 (link) were generated as previously described.75 (link) Approximately 50–100 μg of HEK293T gDNA was used to generate circles for each reaction. Using a Diagenode Bioruptor XL sonicator at 4°C, gDNA was sonicated to an average size of approximately 50 bp, with a visible range of 200–1,000 bp, as determined by agarose gel electrophoresis. The enzymatic procedure to generate circles was carried out as previously described.49 (link) For the in vitro digest of the circles, gRNAs were synthesized from IDT and SaCas9 was purchased from Applied Biological Materials. Library production was carried out as previously described for CHANGE-seq.50 (link) Libraries were quantified by the qPCR-based KAPA Library Quantification Kit (KAPA Biosystems), pooled, and sequenced with 150-bp paired-end reads on an Illumina NextSeq instrument. Read counts were obtained using previously described methods and software for CHANGE-seq.50 (link) The following parameters were used for running the analysis pipeline: read threshold of 4; window size of 3; mapq threshold of 50; start threshold of 1; gap threshold of 3; mismatch threshold of 6; and PAM of NNGRRN.43 (link)
Bioruptor xl
Manufactured by Diagenode
Sourced in Belgium
The Bioruptor XL is a laboratory instrument designed for the shearing and fragmentation of DNA and chromatin samples. It utilizes powerful ultrasound waves to generate shear forces that break down the samples into smaller fragments. The Bioruptor XL is capable of processing multiple samples simultaneously, making it a versatile tool for various applications in molecular biology and genomics research.
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Lab products found in correlation
C jun ap 1, by Abcam (2 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (2 mentions)
Nextseq instrument, by Illumina (1 mentions)
Kapa library quantification kit, by Roche (1 mentions)
M220 focused ultrasonicator, by Covaris (1 mentions)
Prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Thermo Fisher Scientific (1 mentions)
Simplechip plus sonication chromatin ip kit, by Cell Signaling Technology (1 mentions)
Sensifast sybr hi rox kit, by Meridian Bioscience (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Anti trimethyl h3 k9 k27 ab, by Abcam (1 mentions)
11 protocols using bioruptor xl
1
CIRCLE-seq for genome-wide CRISPR off-target analysis
2
Shotgun Metagenome Sequencing Library Preparation
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Shotgun metagenome data were prepared with genomic DNA using various methods. Libraries were built on six samples using a NEBNext protocol, during which metagenomic DNA was fragmented to an average length of ≈350 bp using the Bioruptor XL (Diagenode, Inc.), with the profile of eight cycles of 15 s of sonication and 90 s of rest. Sheared DNA was converted to BGIseq sequencing technology compatible libraries using NEBNext library kit E6070L (New England Biolabs) and blunt‐ended BGISEQ‐500‐compatible adapters AD1 and AD2.[100 ] For all other samples (n = 45) metagenomic data were prepared using the BEST single‐tube library preparation protocol[101 ] as optimized to be BGISEQ‐500 compatible.[100 ] Briefly, genomic DNA was fragmented to 350 bp using an M220 Focused Ultrasonicator (Covaris, Woburn, MA). Sheared DNA was converted into BGISEQ‐500 libraries following four steps: blunt end‐repair, adapter ligation (2 μL of 10 × 10‐6m BGI 2.0 adapters), fill‐in reaction, and SPRI magnetic bead purification (Sigma‐Aldrich). Indexing PCR cycle numbers for all metagenomic libraries (4–14 cycles) were determined through qPCR library quantification. Libraries were pooled equimolar over six lanes in 100bp or 150 bp paired‐end mode on the BGISeq‐500 platform aiming for a minimum of 50 million reads per sample.
3
Two-Step ChIP-Seq Procedure
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For re-ChIP experiments43 (link), ~5e7 cells were used for the first ChIP and chromatin in RIPA buffer was sheared to a median fragment size of 250 bp using a Bioruptor XL (Diagenode). The first ChIP was performed by incubation of sheared chromatin with corresponding antibody-linked magnetic beads at 4 °C overnight. Samples were washed three times with Re-ChIP wash buffer (50 mM Tris-HCl pH 8.0, 500 mM NaCl, 0.1% SDS, 1% NP-40, 2 mM EDTA) and then chromatin-antibody complexes were eluted in Re-ChIP elution buffer (10 mM Tris-HCl pH 8.0, 2% SDS, 15 mM DTT) at 37 °C for 30 min and subsequently diluted 1/20 in ChIP dilution buffer (16.7 mM Tris-HCl pH 8.0, 167 mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS). The second antibody-linked-magnetic beads were added and incubated overnight on a rotary shaker at 4 °C. The second-round chromatin-antibody complexes were captured, washed, and eluted similarly and then DNA was purified for qPCR as above.
4
ChIP-qPCR Evaluation of p53 Binding
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ChIP assays were performed as described [11 (link)] in triplicate. Briefly, after treatment, the cellular material was cross-linked with methanol free 1% formaldehyde (Sigma). Then, cell lysates were sonicated using conditions that yield 200-500 bp DNA fragments using a Bioruptor XL (Diagenode, Denville, NJ). DNA-protein complexes were immunoprecipitated with 1 μg of DO-1 p53-specific monoclonal antibody per condition. Mouse Ig (Santa Cruz Biotechnology) was used as a negative control. qPCR was performed on immunoprecipitated chromatin to determine p53 enrichment occupancy on TLR3 and p21 promoter regions. Amplification of GADPH promoter region was used as a negative control. ChIP primers for TLR3, p21 and GADPH were previously described [23 (link)–24 (link)]. qPCR and melting curve analysis was performed using the SYBR® Green (Invitrogen) dye detection method on the ABI PRISM 7900 HT Sequence Detection System under default conditions. Enrichment in the ChIP samples was calculated as a fraction of the Input (%).
5
Isolation of Cytosolic and Nuclear Fractions from Human Breast Cancer Cells
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Human breast cancer CAL51 wild-type (WT) and MUS81 knockout (KO) cell lines were cultured in DMEM + 10% FBS, supplemented with L-Glutamine and Penicillin–Streptomycin until confluency. Cells were washed twice with cold PBS, scraped from the flask surface and resuspended in PBS. After spinning the cells down at 4ºC, 1500 RPM for 3 min, PBS was removed and the cells were resuspended in buffer A (20 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, protease inhibitors). After the addition of Nonidet® P40 (substitute) to 0.4%, the cells were incubated for 20 min in ice. Following centrifugation at 4ºC and 1500 RPM for 8 min, the supernatant corresponding to the cytosolic fraction was removed. The pellet was resuspended in buffer B (20 mM Tris pH 7.5, 3 mM MgCl2, 410 mM NaCl, 10% glycerol, 1% Triton-X, protease inhibitors). Sonication was then performed in Bioruptor XL (Diagenode) set to 15 × [15 s + 35 s rest]. After centrifugation at maximal speed, the supernatant, now the nuclear extract, was recovered, aliquoted and stored at −80 °C. Protein concentration was measured by Bradford assay.
6
Chromatin Immunoprecipitation (ChIP) in A549 Cells
7
Isolation of Hemozoin from Malaria Parasites
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Lysed iRBC were incubated with a final concentration of 0.1% saponin for 2 min at RT. The samples were then sonicated twice using the Bioruptor® XL (Diagenode, Denville, NJ) for 30 s on high speed, then spun down at 11,000 × g for 30 min at RT. Following this, the pellet was washed thrice with 100 mM NaHCO3, 2% SDS (pH 9), followed by centrifugation (11,000×g, 2 min, RT). The washed pellet was sonicated again for 30 s at high speed. The lysate was centrifuged at 11,000×g for 30 min at RT then washed twice with 2% SDS (pH 9), then twice again with purified or Milli-Q water. The isolated Hz was resuspended in distilled water and stored at − 80 °C.
8
Chromatin Immunoprecipitation Protocol
9
ChIP Assay for H3K4me3 Enrichment
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Chromatin immunoprecipitation (ChIP) assays were performed as described previously50 (link). Briefly, monocytes (5 × 106) were treated with either uRBC or iRBC for 4 h before crosslinking protein-DNA complexes with 1% formaldehyde for 10 min at room temperature. ChIP was performed using a SimpleChIP Plus Sonication Chromatin IP Kit (Cell Signaling Technologies) according to the manufacturer’s instructions. DNA was sheared with Bioruptor XL (Diagenode) with a HI pulse setting and 30 s on and 30 s off pulses. The cycle was repeated 30 times, resulting in a sonication time of 30 min in total to achieve chromatin fragments of 200 to 1,200 base pairs. Chromatin immunoprecipitation was performed with 1 μg of anti-H3K4me3 or anti-histone H3 antibodies (Millipore), followed by reversal of cross-linking. Immunoprecipitated DNA was purified with the MinElute PCR purification kit (QIAGEN). The quantitative PCR reaction was then performed on the immunoprecipitated DNA fragments with a SensiFAST SYBR Hi-ROX Kit (Bioline) and the following primer pairs: IL-6, forward 5′-AGCTCTATCTCCCCTCCAGG-3′ and reverse 5′-ACACCCCTCCCTCACACAG-3′; TNF, forward 5′- CAGGCAGGTTCTCTTCCTCT-3′ and reverse 5′- GCTTTCAGTGCTCATGGTGT-3′16 (link). Enrichment of H3Kme3 in the promoter regions of the IL-6 and TNF genes was expressed as a percentage of the input DNA.
10
Chromatin Immunoprecipitation Protocol
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Chromatin (approximately 5 × 106 cells) was sonicated (Bioruptor XL [Diagenode, Denville, NJ]) prior to immunoprecipitation with rabbit antibodies: c-Jun (AP-1) (Abcam, Cambridge, MA; Cat. # ab31419) and IgG (Millipore, Billerica, MA; Cat. # 12-370) antibodies bound to Dynabeads Protein A (Life Technologies). ChIP Primers are listed in Table S4 .
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