Genomic DNA was extracted from bone marrow using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands). Approximately 1.5 μg of genomic DNA was fragmented to segments between 150 and 250 bp in length using the Bioruptor Pico Sonication System (Diagenode, Belgium). The resulting DNA was then end-repaired and ligated to Illumina adapters (Illumina, San Diego, CA, USA). Sequence indexes were added to the samples to allow all samples to be sequenced in a single flow cell. Small fragments of ~100 bp and unligated adapters were removed using the AMPure purification system (Agencourt Bioscience, Beverly, MA, USA). Sequencing libraries were then hybridized with the capture probes. Streptavidin-coated paramagnetic beads were used to remove unbound DNA. The captured DNA was finally eluted from the magnetic beads by digestion of the cRNA capture probes and purified. The enriched DNA was then amplified using universal primers targeting the paired-end adapters, clusters were generated, and DNA was sequenced on a NextSeq 550 instrument (Illumina) with 2×151 bp reads. All procedures were performed according to the manufacturer’s instructions.
Bioruptor pico sonication system
Manufactured by Diagenode
Sourced in Belgium
The Bioruptor Pico Sonication System is a compact and high-performance device designed for efficient DNA/chromatin shearing. It utilizes focused acoustic energy to fragment samples, enabling consistent and reproducible sample preparation for various downstream applications such as ChIP-seq, ATAC-seq, and DNA methylation analysis.
Automatically generated - may contain errors
Lab products found in correlation
Adapters, by Illumina (4 mentions)
Qiaamp dna blood mini kit, by Qiagen (4 mentions)
Nextseq 550, by Illumina (3 mentions)
Qiaquick pcr purification kit, by Qiagen (2 mentions)
Maxwell rsc dna ffpe kit, by Promega (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Thruplex dna seq kit, by Takara Bio (1 mentions)
Hiseq 2500 sequencer, by Illumina (1 mentions)
H3k4me3 antibody, by Merck Group (1 mentions)
Pcr purification kit, by Qiagen (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Miseq sequencing system, by Illumina (1 mentions)
Miseq reagent kit, by Illumina (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Bst polymerase, by New England Biolabs (1 mentions)
Herculase 2 fusion polymerase, by Agilent Technologies (1 mentions)
T4 ligase, by New England Biolabs (1 mentions)
Nextseq 550 instrument, by Illumina (1 mentions)
Trimethyl histone h3 lys 9, by Merck Group (1 mentions)
Acetyl histone h3 lys9, by Merck Group (1 mentions)
14 protocols using bioruptor pico sonication system
1
Targeted Sequencing of Genomic DNA from Bone Marrow
2
Chromatin Immunoprecipitation of T-bet in Ifng Locus
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A total of 5 × 106 CD4 cells from infected and uninfected mice was cross-linked with 1% formaldehyde for 10 min at room temperature. After quenching of formaldehyde with 125 mM glycine, chromatin was sheared by sonication using a Bioruptor Pico sonication system (Diagenode). The fragmented chromatin was ∼200–1000 bp, as analyzed on agarose gels. After preclearing, chromatin (500 μg) was subjected to immunoprecipitation with specific anti–T-bet Ab (H-210; Santa Cruz Biotechnology), or with anti-Ig as negative control, at 4°C overnight. Immunocomplexes were recovered by incubation with Protein G beads. DNA was purified using a QIAquick PCR Purification Kit (QIAGEN) and used as a template for PCR amplification, with specific primers flanking the T-bet binding sites in the CNS enhancer regions of the Ifng locus, as defined previously (18 (link), 19 (link)). The following primers (20 (link)) were used: CNS-34 (−33,884 to −33,757): sense 5′-TTTGGTTGGCTTACTCACTTTATTC-3′ and antisense 5′-CATCGAGGTGCCATTAACTATCA-3′; CNS-22 (−21,836 to −21,724): sense 5′-GGTGATCCACAGGAAGGAGA-3′ and antisense 5′-GAGCAGAAATTTGGCCTCTT-3′; and CNS+30 (+29,686 to +29,755): sense 5′-TCCGACGAGTGACCAAGATG-3′ and antisense 5′-CCCCAGCGGCTCTCTAAAG-3′.
Chromatin immunoprecipitation data are presented as the percentage of input DNA.
Chromatin immunoprecipitation data are presented as the percentage of input DNA.
3
ChIP-seq analysis of H3K4me3 in plasma cells
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As a complementary approach to identify variants located in genomic regions with regulatory activity in plasma cells, we analyzed previously published31 (link) chromatin immunoprecipitation sequencing (ChIP-seq) data for the H3K4me3 histone modification38 (link). Briefly, L363 cells (DSMZ) were cross-linked with 1% paraformaldehyde (ThermoFisher, #28908). DNA was sonicated into 200–400 bp fragments (Bioruptor Pico Sonication System, Diagenode, Belgium). For pull-down, we used 1–10 μg of H3K4me3 antibody (Millipore, #04-745). Fragments were de-cross-linked and purified (Zymogen, #D5205). ChIP-seq libraries were prepared using the ThruPLEX DNA-seq Kit (Rubicon Genomics, #R400406) and sequenced on Illumina HiSeq 2500 sequencer (paired-end; 2 × 125 cycles). De-multiplexing and generation of FASTQ files was performed using bcl2fastq v.1.8. FastQC (v0.11.5)39 (link) was used to assess read quality low-quality bases were removed using Trimmomatic (v.0.36)40 (link),41 (link) prior to alignment. using Bowtie2 (v.2.3.0)41 (link). Coverage in 50 bp over the SOHL2 region was calculated with the GenomicAlignments and GenomicRanges R-packages42 (coverage and binnedAverage functions) and scaled to Counts-per-million (CPM) relative to the total number of reads per library.
4
T-bet ChIP-qPCR on Ifng Locus
5
Chromatin Immunoprecipitation Liver Analysis
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Chromatin Immunoprecipitation (ChIP) analysis was performed using frozen liver samples, as described previously [20 (link),28 (link),29 (link)], with minor modifications. Briefly, approximately 100 mg liver samples were crosslinked with 1% formaldehyde for 10 min and halted by 125 mM glycine. Samples were then subjected to lysis using an SDS lysis buffer (1% SDS, 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 0.1% sodium deoxycholate, and 1% Triton-X) with a proteinase inhibitor cocktail on ice for 30 min. The lysates were sonicated with a Bioruptor Pico sonication system (Diagenode, Liège, Belgium) to shear DNAs into 200 to 1000-bp size. The sonication condition was 30 s on and 30 s off for 40 cycles. The fragment size was confirmed by agarose gel electrophoresis. ChIP-grade antibodies against H3K4me3 (9751S, Cell Signaling Technology, Boston, MA, USA) and H3K27me3 (9733S, Cell Signaling Technology) were used for immunoprecipitation. A mouse IgG antibody (12-371B, EMD Millipore) was used as a negative control. The immunoprecipitation and DNA purification procedures were described clearly in the previous study [29 (link)]. The purified DNA was determined by qRT-PCR with specific primers (Supplemental Table S2 ) using an SYBR Premix EX Taq kit (Takara Bio) in the QuantStudio5 fluorescent quantitative PCR instrument (Thermo Scientific Company).
6
Genome-Wide CRISPR Off-Target Profiling
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Human embryonic kidney (HEK) 293T/17 cells (2.5 × 105) were transfected with 500 ng of a SpCas9-expressing plasmid, together with 250 ng of each single-guide RNA–coding plasmid or an empty pUC19 vector (background control), 10 pmol of the bait dsODN (designed according to the original GUIDE-seq protocol), and 50 ng of a pEGFP-IRES-Puro plasmid, expressing both enhanced GFP (EGFP) and the puromycin resistance genes. One day after transfection, cells were replated and selected with puromycin (1 μg/ml) for 48 hours to enrich for transfected cells. Cells were then collected, and genomic DNA was extracted using the DNeasy Blood and Tissue Kit (Qiagen) and sheared to an average length of 500 bp with the Bioruptor Pico Sonication System (Diagenode). Library preparation was performed using the original adapters and primers according to previous work (27 (link)). Libraries were sequenced with a MiSeq sequencing system (Illumina) using an Illumina MiSeq Reagent kit V2-300 cycles (2 × 150-bp paired-end). Raw sequencing data (FASTQ files) were analyzed using the GUIDE-seq computational pipeline (52 (link)). Identified sites were considered bona fide off-targets if a maximum of seven mismatches against the on-target were present and if they were absent in the background control. The GUIDE-seq datasets are available in the BioProject repository under the accession number PRJNA531587.
7
cfDNA Library Preparation Protocol
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The cfDNA was subjected to library preparation as previously described [7 (link)] with modifications. In brief, initially blunt ending and 5′ phosphorylation was performed using T4 polymerase and T4 kinase respectively. Sequencing adaptors were then ligated at both ends using T4 Ligase (New England Biolabs, Ipswich, UK). Nicks were removed in a fill-in reaction using Bst polymerase (New England Biolabs). Library amplification was performed using Herculase II Fusion Polymerase (Agilent Technologies, Santa Clara, CA), and unique barcodes were assigned to all samples. At each step, products were purified using Ampure XP magnetic beads according to manufacturer’s instructions.
Prior to library construction of buccal swab, amniotic fluid and CVS samples, extracted genomic DNA was sheared to an average size of 250 bp using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium). Blunt ending, adaptor ligation and adaptor fill-in reactions were performed without intermediate purification steps. A single purification step was performed following library amplification using Ampure XP magnetic beads according to manufacturer’s instructions.
All library preparation steps were run on Hamilton STAR (Hamilton, Bonaduz, Switzerland) or epMotion (Eppendorf) systems using in-house developed automated methods.
Prior to library construction of buccal swab, amniotic fluid and CVS samples, extracted genomic DNA was sheared to an average size of 250 bp using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium). Blunt ending, adaptor ligation and adaptor fill-in reactions were performed without intermediate purification steps. A single purification step was performed following library amplification using Ampure XP magnetic beads according to manufacturer’s instructions.
All library preparation steps were run on Hamilton STAR (Hamilton, Bonaduz, Switzerland) or epMotion (Eppendorf) systems using in-house developed automated methods.
8
Targeted Sequencing for Variant Analysis
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Genomic DNA was extracted from peripheral blood using a QIAamp DNA Blood Mini Kit (Qiagen, Venlo, The Netherlands). The amount of input DNA was approximately 500 ng. DNA was fragmented into segments between 150 and 250 bp using the Bioruptor® Pico sonication system (Diagenode, Liege, Belgium), end-repaired, and ligated to Illumina adapters (Illumina, San Diego, CA, USA) and indices. Sequencing libraries were hybridized with capture probes (Celemic, Seoul, Korea). The enriched DNA was then amplified, and clusters were generated and sequenced on a NextSeq 550 instrument (Illumina) with 2 × 151 bp reads [17 (link)]. Pathogenicity interpretations of the variants were performed according to the 2015 American College of Medical Genetics and Genomics guidelines by professional medical geneticists, using evidence from variant type assessments, population allele frequency, prediction algorithm results, and searches within databases such as ClinVar.
9
ChIP Analysis of Histone Modifications in YG8JR and Y47JR Mouse Cerebellum
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ChIP analysis of YG8JR and Y47JR mouse tissues (n = 4) was carried out by initial cross-linking of DNA and protein by formaldehyde treatment of homogenized cerebellum tissue samples (45 mg each). DNA was then sheared by sonication using a Bioruptor Pico sonication system (Diagenode), followed by immunoprecipitation with trimethyl-Histone H3 (Lys9) (Millipore Cat# 07-442, RRID:AB_310620 ) or acetyl-Histone H3 (Lys9) (Millipore Cat# 07-352, RRID:AB_310544 ) antibodies. The DNA was reverse-cross linked and extracted by standard phenol/chloroform/glycogen extraction and ethanol precipitation. Quantitative PCR amplification of the co-immunoprecipitated DNA was carried out with Fast SYBR Green in a QuantStudio 7 Flex Real-Time PCR (Applied Biosystems, MA, United States) using three sets of FXN primers (Pro: promoter, Up: upstream, Down: downstream of GAA repeat region) as previously described (Herman et al., 2006 (link); Al-Mahdawi et al., 2008 (link)). Each value of immunoprecipitated DNA was processed in triplicate qPCR analysis. Relative quantification values were identified by 2–ΔΔCt method and were normalized to the input values. For each experiment, minus antibody immunoprecipitation and ddH2O without chromatin samples were used as negative controls.
10
Chromatin Immunoprecipitation (ChIP) Assay
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The chromatin immunoprecipitation assay was performed using a Zymo-Spin ChIP kit (Zymo Research, Irvine, CA, USA, Cat. No. D5210) as previous described10 (link). Chromatin was crosslinked with 1% formaldehyde for 10 min at room temperature. The crosslinking reaction was stopped at a final concentration of 0.125 M glycine. The prepared nuclei were mechanically sheared using the Bioruptor Pico sonication system (Diagenode, Liege, Belgium) at 4 °C in 30 s on/off for 25 cycles. A small portion of the chromatin solution was reserved as input DNA. The chromatin samples were incubated with the indicated antibodies by rotating for 16 h at 4 °C. SureBeads Protein A/G magnetic beads (Bio-Rad, Cat. No. 161-4013) were incubated at 4 °C for 1 h by rotation to elute the antibody-chromatin complexes. The bead-antibody-chromatin complex was washed three times with chromatin wash buffer (Zymo Research, Cat. No. D5210). The eluted chromatin was decrosslinked with 5 M NaCl, and then, the chromatin proteins were degraded with proteinase K (genDEPOT, Katy, TX, USA, Cat. No. P2170). ChIP DNA was purified using a QIAquick Spin column (Qiagen, Hilden, Germany, Cat. No. 28115). The ChIP DNA and input were analyzed via qPCR using primer pairs specific to the target gene promoters. The primer sequences are listed in Supplementary Table 2 .
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