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Dna smart chip seq kit

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The DNA SMART ChIP-Seq Kit is a lab equipment product designed for chromatin immunoprecipitation (ChIP) and subsequent sequencing. It enables the preparation of ChIP-Seq libraries from small amounts of input material.

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Lab products found in correlation

Hiseq 4000, by Illumina (3 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions) Strep tactin xt, by IBA Lifesciences (2 mentions) Truchip chromatin shearing kit, by Covaris (2 mentions) S220 focused ultrasonicator, by Covaris (2 mentions) Kapa hyperplus kit, by Roche (2 mentions) Hiseq 2000 sequencer, by Illumina (2 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Novaseq, by Illumina (1 mentions) Miseq platform, by Illumina (1 mentions) Truseq indexed adapter, by Illumina (1 mentions) Kapa hyper prep kit, by Illumina (1 mentions) Nextseq 500, by Illumina (1 mentions) Dynabeads protein g magnetic beads, by Thermo Fisher Scientific (1 mentions) Hiseq 2000 platform, by Illumina (1 mentions) Nextseq, by Illumina (1 mentions) High sensitivity dna analysis kit, by Agilent Technologies (1 mentions) Tapestation 2200, by Agilent Technologies (1 mentions) Kapa library quantification kit, by Roche (1 mentions) True microchip kit, by Diagenode (1 mentions)

15 protocols using dna smart chip seq kit

1

Extracting DNA for ChIP-Seq Analysis

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To extract DNA, the pull-down chromatin fractions were incubated in de-crosslinking buffer (50 mM Tris–HCl pH 8.0, 200 μg/mL Protease K, 2% SDS) overnight at 65 °C. An equal volume of phenol: chloroform: isoamyl alcohol (25:24:1,v/v) (311-90151, Wako) was added, vortexed and then centrifuge at 16,000g for 5 min at room temperature. The upper aqueous phase was transferred to a new tube. This step was repeated. One-tenth volume of 3 M sodium acetate, glycogen, and the double volume of ethanol were added and incubated for 2 h at − 80 °C. After centrifugation at 20,000g for 30 min at 4 °C, the pellet was retained, washed with 70% ethanol and dried. The dried pellet was dissolved in 10 μg/mL RNase A in DNA dilution buffer attached to DNA SMART ChIP-Seq Kit (634865, Takara). After incubating for 1 h at 37 °C, DNA was purified by phenol–chloroform extraction and ethanol precipitation to remove RNase A. DNA samples were kept at − 30 °C until use. DNA concentrations of each sample were measured by Qubit. cDNA libraries were synthesized by DNA SMART ChIP-Seq Kit (634865, Takara). The size distributions of the libraries were checked by an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit. Pooled amplicon library was sequenced on the Illumina MiSeq platform with paired-end 2 × 250 bp reads for HeLa1.3 and U2-OS cells, and with single-end 75 bp reads for MEL cells.
Ide S., Sasaki A., Kawamoto Y., Bando T., Sugiyama H, & Maeshima K. (2021). Telomere-specific chromatin capture using a pyrrole–imidazole polyamide probe for the identification of proteins and non-coding RNAs. Epigenetics & Chromatin, 14, 46.
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2

Genome-Wide Mapping of RORα and RORγt

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RORα-TS and RORγt ChIP-Seq was performed as described (Ciofani et al., 2012 (link)) with the following modifications. For each ChIP, 20–80 million cells were cross-linked with paraformaldehyde; chromatin was isolated using truChIP Chromatin Shearing Kit (Covaris) and fragmented with a S220 Focused-ultrasonicator (Covaris). Twin-strep (TS) tagged RORα protein was precipitated using Strep-TactinXT according to the manufacturer’s protocol (IBA Lifesciences). Following immunoprecipitation, the protein-DNA crosslinks were reversed and DNA was purified. DNA from control samples was prepared similarly but without immunoprecipitation. Sequencing libraries were made from the resulting DNA fragments for both ChIP and controls using DNA SMART™ ChIP-Seq Kit (Takara) for RORα-TS ChIP-Seq and KAPA HyperPlus Kit (Roche) for RORγt ChIP-Seq. The ChIP-Seq libraries were sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000.
Hall J.A., Pokrovskii M., Kroehling L., Kim B.R., Kim S.Y., Wu L., Lee J.Y, & Littman D.R. (2022). Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element. Immunity, 55(11), 2027-2043.e9.
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3

High-Purity DNA Extraction and Sequencing

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To extract high-purity DNA, input sample (1 ng) and isolated DNA samples (Cy3-labeled and nonlabeled DNAs) were added to an equal volume of phenol:chloroform:isoamylalcohol (25:24:1, v/v) and centrifuged at 16,000g at room temperature for 7 min. The upper aqueous phase was transferred to new tube. One-tenth volume of 3 M sodium acetate, glycogen, and double volume of 100% ethanol were added and incubated at −80°C for 1 hour. The pellets were retained after centrifugation at 20,000g for 30 min at 4°C and washed with 70% ethanol, dried and dissolved in Milli-Q water, and stored at −30°C until use. Complementary DNA (cDNA) libraries were synthesized by the DNA SMART ChIP-seq kit (#634865; Takara) from half of input, Cy3-labeled and nonlabeled samples. The size distribution of the libraries was checked by an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit. Pooled amplicon library was sequenced with paired-end 2 × 100 bp reads on the Illumina HiSeq and NovaSeq platforms.
Nozaki T., Shinkai S., Ide S., Higashi K., Tamura S., Shimazoe M.A., Nakagawa M., Suzuki Y., Okada Y., Sasai M., Onami S., Kurokawa K., Iida S, & Maeshima K. (2023). Condensed but liquid-like domain organization of active chromatin regions in living human cells. Science Advances, 9(14), eadf1488.
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4

Genome-Wide Mapping of RORα and RORγt

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RORα-TS and RORγt ChIP-Seq was performed as described (Ciofani et al., 2012 (link)) with the following modifications. For each ChIP, 20–80 million cells were cross-linked with paraformaldehyde; chromatin was isolated using truChIP Chromatin Shearing Kit (Covaris) and fragmented with a S220 Focused-ultrasonicator (Covaris). Twin-strep (TS) tagged RORα protein was precipitated using Strep-TactinXT according to the manufacturer’s protocol (IBA Lifesciences). Following immunoprecipitation, the protein-DNA crosslinks were reversed and DNA was purified. DNA from control samples was prepared similarly but without immunoprecipitation. Sequencing libraries were made from the resulting DNA fragments for both ChIP and controls using DNA SMART™ ChIP-Seq Kit (Takara) for RORα-TS ChIP-Seq and KAPA HyperPlus Kit (Roche) for RORγt ChIP-Seq. The ChIP-Seq libraries were sequenced with paired-end 50 bp reads on an Illumina HiSeq 4000.
Hall J.A., Pokrovskii M., Kroehling L., Kim B.R., Kim S.Y., Wu L., Lee J.Y, & Littman D.R. (2022). Transcription factor RORα enforces stability of the Th17 cell effector program by binding to a Rorc cis-regulatory element. Immunity, 55(11), 2027-2043.e9.
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5

ChIP-seq Library Preparation and Sequencing

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10–50 ng ChIP DNA was used for library construction using KAPA HyperPrep kit and Illumina TruSeq indexed adapters. Dual size selection was performed after 18 cycles of PCR amplification according to KAPA’s recommended protocol. 12-plex, single-indexed, 1×75 bp Illumina NextSeq500 sequencing was employed for ChIPseq libraries to yield an average of 3.5–4.0 × 107 reads per sample.
Library preparation and sequencing for H3K4me3 ChIPseq was performed by the UNC High Throughput Sequencing Facility. H3K4me1 ChIPseq libraries were constructed using DNA SMART ChIP Seq kit (Clontech) with 10 ng ChIP DNA, 18 cycles of amplification, and double size selection post-PCR following the manufacturer’s suggested protocol. For the DNA SMART ChIPseq libraries, the first three bases of the sequencing read, corresponding to the template switching oligo, were trimmed prior to mapping.
Raw and processed ChIPseq data is deposited in GEO as SuperSeries GSE87424. Python code generated in the laboratory for ChIPseq analysis is available at GitHub (55 ).
Zawistowski J.S., Bevill S.M., Goulet D.R., Stuhlmiller T.J., Beltran A.S., Olivares-Quintero J.F., Singh D., Sciaky N., Parker J.S., Rashid N.U., Chen X., Duncan J.S., Whittle M.C., Angus S.P., Velarde S.H., Golitz B.T., He X., Santos C., Darr D.B., Gallagher K., Graves L.M., Perou C.M., Carey L.A., Earp H.S, & Johnson G.L. (2017). Enhancer Remodeling During Adaptive Bypass to MEK Inhibition Is Attenuated by Pharmacological Targeting of the P-TEFb Complex. Cancer discovery, 7(3), 302-321.
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6

ChIP-seq protocol for H2A.Z and H3K36me3

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Chromatin isolation and subsequent ChIP of WT T87 cells were performed according to the published method (Satoh et al. 2020 ) with modifications, as follows. Fixed cells (0.2 g) were used for chromatin isolation. ChIP was performed with 10–20 ng of solubilized chromatin, Dynabeads Protein-G magnetic beads (Invitrogen) and antibodies: 2.4 µg of an anti-H2A.Z rabbit polyclonal antibody (Kudo et al. 2020 ) and 1.0 µg of an anti-H3K36me3 rabbit polyclonal antibody (Abcam: ab9050) were used in this experiment. Successful enrichment of ChIPed DNA was validated by quantitative PCR (qPCR) according to Deal et al. (2007) (link) for H2A.Z, and to Yang et al. (2014) (link) for H3K36me3. ChIP-seq libraries were prepared using a DNA SMART ChIP-seq Kit (Clontech) with 1.0 ng of ChIPed DNA and input DNA (DNA extracted from sheared chromatin), respectively. Libraries were size selected at 200–400 bp using AMPure beads. NGS was performed using a 51 bp single-ended protocol on an Illumina HiSeq 2000 platform.
Hata T., Satoh S., Takada N., Matsuo M, & Obokata J. (2021). Kozak Sequence Acts as a Negative Regulator for De Novo Transcription Initiation of Newborn Coding Sequences in the Plant Genome. Molecular Biology and Evolution, 38(7), 2791-2803.
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7

ChIP-seq Analysis of H3K9me2 in Arteriosclerosis

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ChIP was performed as described in the Methods in the online-only Data Supplement. For genome-wide analysis, the DNA SMART ChIP-seq kit (Clontech, 634865) was used to generate Illumina-compatible sequencing libraries from 100 pg to 2 ng of DNA from 2 independent H3K9me2 ChIP experiments and associated input. Libraries were sequenced (Illumina NextSeq) using paired-end 75 bp reads. ChIP-seq reads were trimmed using Cutadapt v1.9,37 aligned to the mouse GRCm38 genome using Bowtie2 v2.2,38 (link) and reads per gene promoter (within ±1 kb of the transcription start sites) quantified using SeqMonk v1.4 (http://www.bioinformatics.babraham.ac.uk/projects/seqmonk). Genes showing fewer than 20 read counts in the input samples were removed from further analysis and the ratio of H3K9me2/input signal was computed. Of genes with H3K9me2/input ratios in the top 25th percentile, 63 genes were associated with arteriosclerosis according to the Cardiovascular Disease Portal39 (link) (https://rgd.mcw.edu/rgdCuration/?module=portal&func=show&name=cardio).
Harman J.L., Dobnikar L., Chappell J., Stokell B.G., Dalby A., Foote K., Finigan A., Freire-Pritchett P., Taylor A.L., Worssam M.D., Madsen R.R., Loche E., Uryga A., Bennett M.R, & Jørgensen H.F. (2019). Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling. Arteriosclerosis, Thrombosis, and Vascular Biology, 39(11), 2289-2302.
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8

MeDIP Sequencing Library Preparation

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Sequencing libraries were prepared by DNA SMART™ ChIP-Seq kit (634865, Clontech). This kit generates sequencing libraries from low-input ssDNA MeDIP samples (100 pg - 10 ng) without using ligation. Sequencing libraries were amplified by PCR using primers containing Illumina adapters. Amplification cycles used for 0.5, 5, 10, 100 ng GM12878 DNA samples were 17, 13, 13, 12 cycles, respectively. Amplification cycles used for 10 ng mouse DNA samples were 13 cycles. The libraries were purified using Ampure XP beads (A63880, Beckman Coulter). The library fragment size was determined using high sensitivity DNA analysis kit (5067–4626, Agilent) on an Agilent 2200 TapeStation. Sonicated DNA for MeDIP had average fragment size around 200 bp (Supplementary Fig. S1a). After library preparation, the average peak size shifted to 350 bp as library construction added about 150 bp adaptor (75 bp on each end) to the MeDIP DNA (Supplementary Fig. S1b). KAPA library quantification kit (KK4809, Kapa Biosystems) was used to determine effective library concentrations. The final concentrations of libraries submitted for sequencing were ~8 nM (20 μl and 37 ng DNA). The libraries were sequenced on an Illumina HiSeq 4000 with single-end 50 nt read. Typically 15–20 million reads were generated per library.
Zhu Y., Cao Z, & Lu C. (2019). Microfluidic MeDIP-seq for low-input methylomic analysis of mammary tumorigenesis in mice. The Analyst, 144(6), 1904-1915.
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9

H3K4me1 ChIP-seq in SUM159 Cells

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The H3K4me1 ChIP-seq was obtained with 10 million SUM159 cells. H3K4me1 ChIP-seq libraries were constructed using the DNA SMART ChIP-seq Kit (Clontech) with 10 ng ChIP DNA (NCBI GEO: GSE87424) [16 (link)]. Raw fastq sequences were downloaded from the GEO and processed with the same methods as the original study.
Zhao D, & Zheng D. (2018). SMARTcleaner: identify and clean off-target signals in SMART ChIP-seq analysis. BMC Bioinformatics, 19, 544.
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10

OLIG2 ChIP-Seq Protocol

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OLIG2 Low-cell ChIP was performed using the True MicroChIP Kit (Diagenode) and libraries were constructed with the DNA SMART ChIP-Seq Kit (Clontech) by following the manufacturer’s instructions. OLIG2 antibody used for ChIP-Seq was purchased from EMD Millipore (AB9610). Rabbit IgG unconjugated polyclonal antibody used for ChIP-Seq was purchased from Cell Signaling Technology (2729S). EZH2 antibody was purchased from EMD Millipore (17-662). SUZ12 antibody was purchased from Abcam (ab12073). Mouse IgG antibody was purchased from EMD Millipore (12-371).
Wei H., Dong X., You Y., Hai B., Duran R.C., Wu X., Kharas N, & Wu J.Q. (2021). OLIG2 regulates lncRNAs and its own expression during oligodendrocyte lineage formation. BMC Biology, 19, 132.
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