Chirp was performed as described by Chu et al 18 . Briefly, mouse livers were cross-linked using glutaraldehyde. After glycine quenching, the nuclear lysate was sonicated for 25–30 cycles 30 seconds on 30 seconds off at 4°C with BioRuptor twin sonicator (Diagenode). LeXis and LacZ pulldown probes with BiotinTEG at 3’ were designed by Biosearch Tecnologies (see supplementary information ) and allowed to hybridize overnight with sonicated chromatin at 37 °C (100 pmol probe per 1 mL chromatin). Following hybridization, C1 Dynabeads (Life Technologies) were added and incubated for 30 minutes. For protein elution for mass spectrometry analysis, washed beads were resuspended in 3× original volume of DNase buffer (100 mM NaCl and 0.1% NP-40), and protein was eluted with a cocktail of 50 mM triethyl ammonium bicarbonate, 12 mM sodium lauryl sarcosine, and 0.5% sodium deoxycholate supplemented with 100 ug/ml RNase A (Sigma-Aldrich) and 0.1 Units/microliter RNase H (Epicenter), and 100 U/ml DNase I (Invitrogen). For RNA isolation, beads were resuspended in protensase K buffer (100 mM NaCl, 10 mM TrisCl pH 7.0, 1 mM EDTA, 0.5% SDS, 5% by volume Proteainse K Ambion AM2546 20 mg/ml) and incubated at 50 °C followed by Trizol isolation and DNase treatment.
Dynabeads c1
Manufactured by Thermo Fisher Scientific
Sourced in United States
Dynabeads C1 are superparamagnetic beads designed for a variety of biological applications. They feature a uniform size distribution and a high magnetic content, allowing for efficient separation and purification of target molecules or cells.
Automatically generated - may contain errors
Lab products found in correlation
Rnaseh, by Illumina (2 mentions)
Bioruptor twin sonicator, by Diagenode (2 mentions)
Rnase a, by Merck Group (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
Lds buffer, by Thermo Fisher Scientific (1 mentions)
Thermomixer, by Eppendorf (1 mentions)
Protein assay dye reagent, by Bio-Rad (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Onetouch 2, by Thermo Fisher Scientific (1 mentions)
Ion torrent personal genome machine, by Thermo Fisher Scientific (1 mentions)
5 protocols using dynabeads c1
1
Chromatin Interaction Capture by Hybridization
2
Chromatin Interaction Capture by Hybridization
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Chirp was performed as described by Chu et al 18 . Briefly, mouse livers were cross-linked using glutaraldehyde. After glycine quenching, the nuclear lysate was sonicated for 25–30 cycles 30 seconds on 30 seconds off at 4°C with BioRuptor twin sonicator (Diagenode). LeXis and LacZ pulldown probes with BiotinTEG at 3’ were designed by Biosearch Tecnologies (see supplementary information ) and allowed to hybridize overnight with sonicated chromatin at 37 °C (100 pmol probe per 1 mL chromatin). Following hybridization, C1 Dynabeads (Life Technologies) were added and incubated for 30 minutes. For protein elution for mass spectrometry analysis, washed beads were resuspended in 3× original volume of DNase buffer (100 mM NaCl and 0.1% NP-40), and protein was eluted with a cocktail of 50 mM triethyl ammonium bicarbonate, 12 mM sodium lauryl sarcosine, and 0.5% sodium deoxycholate supplemented with 100 ug/ml RNase A (Sigma-Aldrich) and 0.1 Units/microliter RNase H (Epicenter), and 100 U/ml DNase I (Invitrogen). For RNA isolation, beads were resuspended in protensase K buffer (100 mM NaCl, 10 mM TrisCl pH 7.0, 1 mM EDTA, 0.5% SDS, 5% by volume Proteainse K Ambion AM2546 20 mg/ml) and incubated at 50 °C followed by Trizol isolation and DNase treatment.
3
Affinity-Based Protein Isolation Protocol
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For each pulldown, 25 µg S1-tagged RNA was immobilized on 50 µL paramagnetic streptavidin beads (Dynabeads C1, Thermo Fisher Scientific, Waltham, MA, USA) in RNA binding buffer (100 mM NaCl, 50 mM HEPES–KOH pH 7.6, 0.1% IGEPAL CA-630, 10 mM MgCl2, 1 µM Pepstatin A, 1 µg/mL Leupeptin, and 1 mM PMSF) at 4 °C for 30 min on a rotation wheel. After two washing steps with RNA binding buffer to remove excess unbound RNA, the prepared beads were incubated with 400 µg nuclear extract and 20 µg yeast tRNA (Thermo Fisher Scientific, Waltham, MA, USA) as competitor in RNA wash buffer (250 mM NaCl, 50 mM HEPES–KOH pH 7.6, 0.1% IGEPAL CA-630, 10 mM MgCl2, 1 µM Pepstatin A, 1 µg/mL Leupeptin, and 1 mM PMSF) for 30 min and 4 °C on a rotation wheel. Unbound proteins were removed by three washing steps with 200 µL RNA washing buffer and bound proteins eluted by heating beads in 1× LDS Buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 100 mM DTT for 10 min at 70 °C and 1400 rpm in a thermomixer (Eppendorf, Hamburg, Germany).
4
Affinity Purification of Protein Complexes
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Chemically synthesized oligonucleotides (Table S1 ) were ordered HPLC-purified from BioSynthesis (Lewisville) and Metabion (Planegg). For pull-downs, 1 nmol of single-stranded DNA lesion (or nondamaged control) oligonucleotide was annealed with 1 nmol of 5′-biotinylated counterstrand with annealing buffer (20 mM Tris-HCl pH 8.0, 10 mM MgCl2, 100 mM KCl) by first heating to 85°C for 5 min and slowly cooling to RT. The double-stranded oligonucleotides were immobilized on 250 μg streptavidin Dynabeads C1 (Thermo) and incubated with different amounts of protein extract ranging from 200-1,000 μg (200 μg: C. elegans, Z. mays and A. thaliana; 400 μg: HEK293 and HeLa; 500 μg: H. salinarum, T. thermophila; 800 μg: S. cerevisiae and 1,000 μg: B. subtilis, E. coli, S. pombe and T. brucei) in 1x PBB buffer (150 mM NaCl, 50 mM Tris-HCl pH 8.0, 0.5% Igepal CA-630, 5 mM MgCl2 and 1x protease inhibitor cocktail [Roche]) rotating at 4°C for 90 min. Protein concentrations were determined using Protein Assay Dye Reagent (Bio-Rad). All samples were prepared in quadruplicate. After incubation, unbound proteins were removed by 3 washes with PBB buffer. The Dynabeads were ultimately resuspended in 25 μl 1x LDS (Thermo) containing 100 mM DTT (Sigma) and heated to 70°C for 10 min.
5
Ion Torrent PGM Barcoded Library Sequencing
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Multiplex barcoded libraries were combined with ion spheres for clonal amplification during emulsion PCR (60 cycles, PGM OT2 200 bp kit, One Touch 2, Thermo-Fisher). Ion spheres were recovered and stained with Alexa Fluor 488 or 647 for Qubit assessment of beads containing template (20-36% polyclonal templates). Libraries were enriched using biotin labeled primers bound to streptavidincoated, metallic beads (DynaBeads C1, Thermo Fisher). Ion spheres were stripped from the streptavidin beads (125 mM NaOH; 0.1% Tween 20) and sequencing was performed on the Ion Torrent Personal Genome Machine (PGM, Thermo-Fisher) using the 200 bp sequencing kit v2 (318v1 chip, pH 7.55, 500 flows). The torrent software suite (Torrent Suite v. 4.0) parsed barcoded reads, assessed read quality, performed trimming and aligned base calls to HG19 using the TMAP aligner (Torrent Mapping Alignment Program) to generate BAM (binary format) files.
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