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Truseq chip library prep kit

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The TruSeq ChIP Library Prep Kit is a laboratory equipment product designed for the preparation of DNA libraries for chromatin immunoprecipitation (ChIP) sequencing. The kit provides the necessary reagents and protocols to convert DNA samples into indexed libraries suitable for sequencing on Illumina platforms.

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Lab products found in correlation

Chip it high sensitivity kit, by Active Motif (3 mentions) Drosophila chromatin, by Active Motif (3 mentions) Epishear probe sonicator, by Active Motif (2 mentions) Antibody against drosophila specific histone variant h2av, by Active Motif (2 mentions) Hiseq 2500, by Illumina (1 mentions) H3k9ac, by Merck Group (1 mentions) H3k27me3, by Merck Group (1 mentions) H3k4me1, by Active Motif (1 mentions) Lamin a c, by Santa Cruz Biotechnology (1 mentions) H3k27ac, by Active Motif (1 mentions) H3k36me3, by Abcam (1 mentions) H3k4me3, by Abcam (1 mentions) Hiseq 4000 sequencer, by Illumina (1 mentions) Bioanalyzer system, by Illumina (1 mentions) Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions) Ultrasonicator, by Covaris (1 mentions) Epishear sonicator, by Active Motif (1 mentions) Hiseq 4000, by Illumina (1 mentions) M220 focused ultrasonicator, by Covaris (1 mentions) Anti h3k27ac, by Active Motif (1 mentions)

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TruSeq kits (44 citations) TruSeq ChIP kit (8 citations)

15 protocols using truseq chip library prep kit

1

Chromatin Immunoprecipitation Sequencing Protocol

Cited 5 times
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ChIP assays were performed as previously described17 (link),18 (link),63 (link),64 (link) for the following histone marks: H3K4me3 (Abcam, #ab8580), H3K4me1 (Active Motif, #39297), H3K36me3 (Abcam, #ab9050), H3K27ac (Active Motif, #39133), H2AZac (Abcam, #ab18262), H3K9ac (Millipore, #06-599) and H3K27me3 (Millipore, #07-449). ChIP of H3K9me3 (Diagenode, #C15500003) was performed as previously described65 (link). We performed lamin ChIP assays in PrEC and LNCaP as previously described66 (link) for both Lamin B1 (Abcam, #ab16048) and Lamin A/C (Santa Cruz, #sc7292). Each ChIP assay was validated by qPCR against an IgG control and enrichment above input. Libraries were prepared with the Illumina TruSeq Chip Library Prep Kit and sequenced on an Illumina HiSeq 2500.
Sequencing data was processed as previously described17 (link),63 (link). Briefly, ChIP-seq reads were aligned to hg19 using bowtie58 (link) (v1.1.0) allowing up to 3 mismatches, discarding ambiguous and clonal reads. All histone ChIP-seq peaks were called using PeakRanger67 (v1.16). Broad domains of lamins (LADs), H3K9me3 and H3K27me3 were called using the enriched domain detector (EDD) for identification of wide genomic enrichment domains68 (link).
Du Q., Bert S.A., Armstrong N.J., Caldon C.E., Song J.Z., Nair S.S., Gould C.M., Luu P.L., Peters T., Khoury A., Qu W., Zotenko E., Stirzaker C, & Clark S.J. (2019). Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer. Nature Communications, 10, 416.
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2

ChIP-seq Analysis of Chromatin Epigenetic Marks

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ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif, Carlsbad, CA), following the manufacturer’s manual. Briefly, formaldehyde-(1%, room temperature, 15 min) fixed chromatin was sheared to fragments of 200–1000 bp with the EpiShear Probe Sonicator (Active Motif). T265 cells were sonicated at 30% amplitude, pulse for 20 s on and 40 s off for a total sonication “on” time of 15 min; ST88-14 and PT2002 cells were sonicated at 30% amplitude, pulse for 20 s on and 40 s off for a total sonication “on” time of 16 min. Sheared chromatin was immunoprecipitated with antibodies against H3K27me3, H3K27ac, H3K4me3 or FOXC1 overnight at 4°C, followed by agarose protein A/G beads enrichment and DNA purification. To date, genome wide FOXC1 binding has not been profiled. A commercially available FOXC1 antibodies was used as it was tested suitable for ChIP-seq assays by ChIP-western blotting and peak calling. Drosophila chromatin (Active Motif) was added to the ChIP reaction and an antibody against Drosophila-specific histone variant H2Av (Active Motif) was included in the pull down. ChIP DNA library was prepared at the CCRSF using TruSeq ChIP Library Prep Kit (Illumina, cat. IP-202-1012) and sequenced on a NextSeq platform for single-end sequencing with a read length of 76 bp. About 25-30 million base-pairs uniquely mapped reads were generated from each sample.
Zhang X., Lou H.E., Gopalan V., Liu Z., Jafarah H.M., Lei H., Jones P., Sayers C.M., Yohe M.E., Chittiboina P., Widemann B.C., Thiele C.J., Kelly M.C., Hannenhalli S, & Shern J.F. (2022). Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor. Cell reports, 40(12), 111363.
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3

DNA Sequencing Library Preparation

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Following DNA purifications, libraries were prepared using the TruSeq ChIP Library Prep Kit according to Illumina protocols, multiplexed library sizes were validated on an Agilent Bioanalyzer system and then sequenced (single-read) on an Illumina HiSeq 4000 sequencer.
Fulton S.L., Wenderski W., Lepack A.E., Eagle A.L., Fanutza T., Bastle R.M., Ramakrishnan A., Hays E.C., Neal A., Bendl J., Farrelly L.A., Al-Kachak A., Lyu Y., Cetin B., Chan J.C., Tran T.N., Neve R.L., Roper R.J., Brennand K.J., Roussos P., Schimenti J.C., Friedman A.K., Shen L., Blitzer R.D., Robison A.J., Crabtree G.R, & Maze I. (2022). Rescue of deficits by Brwd1 copy number restoration in the Ts65Dn mouse model of Down syndrome. Nature Communications, 13, 6384.
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4

Affinity-seq for Identifying Protein-DNA Interactions

Cited 1 time
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Affinity-seq was essentially done as in (24 (link)) with minor adjustments. A ZF array of the protein of interest was amplified then cloned into a universal Affinity-seq vector by recombineering. The resulting construct expressed a fused protein containing 6HisHALO–the 412–511 aa fragment of PRDM9–ZF array of interest. The fused protein was expressed in Rosetta 2 cells at 15°C for 24 h and partially purified by ion exchange chromatography on SP-sepharose. The purified protein was mixed with genomic DNA sheared to ∼200 bp on a Covaris ultrasonicator, and allowed to bind overnight. The protein-DNA complexes were then isolated on HisPur Ni-NTA Resin (Thermo Scientific) preincubated with a partially purified prep of the empty tag to reduce the background. DNA was then eluted and used to prepare genomic libraries using a TruSeq ChIP Library Prep Kit (Illumina). The libraries were sequenced on a HiSeq2500 or NextSeq platform ensuring ∼50 million reads per library. Data were analyzed using a custom pipeline as described previously (24 (link)) and we used the MEME (v4.10.1) software package with default parameters (P-value threshold 0.001 and 150 bp central peaks regions) for motif discovery.
Zuo Z., Billings T., Walker M., Petkov P.M., Fordyce P.M, & Stormo G.D. (2023). On the dependent recognition of some long zinc finger proteins. Nucleic Acids Research, 51(11), 5364-5376.
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5

SNAI2 ChIP-seq Protocol for Gene Regulation

Cited 1 time
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ChIP-seq data used, was published previously (28 (link)) and performed as follows. 1% Formaldehyde-fixed chromatin from RD and SMS-CTR cells were sheared to 200–700 bp with Active Motif EpiShear Sonicator. Chromatin-IP with SNAI2 Ab (CST, Catalogue # 9585) was performed O/N, using ChIP-IT High Sensitivity kit (Active Motif). Drosophila chromatin (Active Motif, Catalogue #53083) and H2Av ab (Active Motif, Catalogue #61686) was used for spike-in normalization across samples. ChIP-seq libraries were prepared using Illumina TruSeq ChIP Library Prep Kit and sequenced on NextSeq500. Reads were mapped to reference genome (version hg19) using BWA. High-confidence ChIP-seq peaks were called by MACS2.1. Raw sequencing data and processed files are available through GEO (GSE137168). For real-time qPCR immunoprecipitated DNA was amplified with BCL2L11 ChIP primers (SNAI2 binding peak 1 & 2) along with negative controls. All signals were normalized against input by the percentage input method. Significance was calculated by Unpaired t test.
Wang L., Hensch N.R., Bondra K., Sreenivas P., Zhao X.R., Chen J., Campos R.M., Baxi K., Vaseva A.V., Sunkel B.D., Gryder B.E., Pomella S., Stanton B.Z., Zheng S., Chen E.Y., Rota R., Khan J., Houghton P.J, & Ignatius M.S. (2021). SNAI2-mediated repression of BIM protects rhabdomyosarcoma from ionizing radiation. Cancer research, 81(21), 5451-5463.
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6

Chromatin Immunoprecipitation with Sonication

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Chromatin immunoprecipitation was performed as described previously (28 ) with the following modifications. Chromatin was sheared in diluted lysis buffer to 200-500bp using a Covaris M220 Focused-Ultrasonicator with the following parameters: 10 minutes, peak incident power 75, duty factor 10%, 200 cycles/burst. Antibodies for ChIP were obtained from commercially available sources: anti-H3K27Ac (Active Motif, #39133), anti-BRD4 (Active Motif, #39909), anti-c-Jun (Cell Signaling, #9165T), anti-c-Fos (Santa Cruz Biotechnology, #sc-166940), anti-BRD3 (Santa Cruz Biotechnology, #sc-515729), and anti-BRD2 (Cell Signaling, #5848S). 5% of the chromatin was not exposed to antibody and was used as control (input). For ChIP-qPCR analysis DNA quantity for each ChIP sample was normalized against input DNA. For ChIP-seq samples, after DNA purification ChIP-seq DNA libraries were prepared with either the TruSeq ChiP Library Prep Kit (Illumina, #IP-202-1012) or the Accel-NGS 2S Plus DNA Library kit (Swift Bioscience, #21024) and sequenced using 75 bp single-end sequencing on an Illumina Hi-seq 4000.
Jameson N.M., Ma J., Benitez J., Izurieta A., Han J.Y., Mendez R., Parisian A, & Furnari F. (2019). Intron 1-Mediated Regulation of EGFR Expression In EGFR-Dependent Malignancies is Mediated by AP-1 and BET Proteins. Molecular cancer research : MCR, 17(11), 2208-2220.
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7

ChIP-seq and In Situ Biotinylation-seq in Drosophila

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Libraries for ChIP-seq and in situ biotinylation-seq were prepared using TruSeq ChIP Library Prep Kit (Illumina, San Diego, CA) and IDT for Illumina-TruSeq RNA UD Index (Illumina). For in situ biotinylation-seq, chromatin from Drosophila melanogaster S2 cells was added to each sample as a spike-in control for normalization. Sequencing reads were acquired using the NovaSeq 6000 or HiSeq 2500 platform. Adapter sequences were trimmed using Trim Galore (v 0.64_dev), and reads were mapped to human genome GRCH38 using the Bowtie2 alignment tool (version 2.3.5.1) with the default settings. The mapped reads from each ChIP-seq and in situ biotinylation-seq dataset were subjected to count per million (CPM) normalization and then used for downstream analysis. IP/input or IP-input enrichment per gene was calculated with R (v. 4.0.2) and its package rtracklayer (version 1.48.0) using the Ensembl GRCH38.92 gene annotation model.
Suzuki H., Abe R., Shimada M., Hirose T., Hirose H., Noguchi K., Ike Y., Yasui N., Furugori K., Yamaguchi Y., Toyoda A., Suzuki Y., Yamamoto T., Saitoh N., Sato S., Tomomori-Sato C., Conaway R.C., Conaway J.W, & Takahashi H. (2022). The 3′ Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies’ association with histone locus bodies. Nature Communications, 13, 2905.
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8

ChIP-seq Analysis of Chromatin Epigenetic Marks

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ChIP was performed using the ChIP-IT High Sensitivity kit (Active Motif, Carlsbad, CA), following the manufacturer’s manual. Briefly, formaldehyde-(1%, room temperature, 15 min) fixed chromatin was sheared to fragments of 200–1000 bp with the EpiShear Probe Sonicator (Active Motif). T265 cells were sonicated at 30% amplitude, pulse for 20 s on and 40 s off for a total sonication “on” time of 15 min; ST88-14 and PT2002 cells were sonicated at 30% amplitude, pulse for 20 s on and 40 s off for a total sonication “on” time of 16 min. Sheared chromatin was immunoprecipitated with antibodies against H3K27me3, H3K27ac, H3K4me3 or FOXC1 overnight at 4°C, followed by agarose protein A/G beads enrichment and DNA purification. To date, genome wide FOXC1 binding has not been profiled. A commercially available FOXC1 antibodies was used as it was tested suitable for ChIP-seq assays by ChIP-western blotting and peak calling. Drosophila chromatin (Active Motif) was added to the ChIP reaction and an antibody against Drosophila-specific histone variant H2Av (Active Motif) was included in the pull down. ChIP DNA library was prepared at the CCRSF using TruSeq ChIP Library Prep Kit (Illumina, cat. IP-202-1012) and sequenced on a NextSeq platform for single-end sequencing with a read length of 76 bp. About 25-30 million base-pairs uniquely mapped reads were generated from each sample.
Zhang X., Lou H.E., Gopalan V., Liu Z., Jafarah H.M., Lei H., Jones P., Sayers C.M., Yohe M.E., Chittiboina P., Widemann B.C., Thiele C.J., Kelly M.C., Hannenhalli S, & Shern J.F. (2022). Single-cell sequencing reveals activation of core transcription factors in PRC2-deficient malignant peripheral nerve sheath tumor. Cell reports, 40(12), 111363.
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9

Nuclei Isolation and DNA Extraction

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Cells (7 × 106 per sample) were scraped, centrifuged and washed with PBS. Cell pellets were resuspended in nuclear extraction buffer (10 mM Tris–HCl pH7.4, 12.5 mM NaCl, 3 mM MgCl, 0.1 mM EDTA, 0.5% IGEPAL) and dounced until nuclei were visible under light microscope with 0.4% Trypan Blue staining. DNase1 (Roche, #04716728001) was added to nuclei pellets of LNCaP (24 U) and PrEC (12 U) and incubated at 37 °C for 15 min. DNase1 reactions were terminated by the addition of 36 mM EDTA and Proteinase K was added before incubating at 55 °C overnight. DNA was purified by phenol-chloroform extraction and ethanol precipitation. Samples were separated using electrophoresis on a 1% agarose gel. 100–300 bp sections were excised and purified using the QIAquick Gel Extraction kit. Libraries were prepared with the Illumina TruSeq Chip Library Prep Kit and sequenced on an Illumina HiSeq 2000.
Du Q., Bert S.A., Armstrong N.J., Caldon C.E., Song J.Z., Nair S.S., Gould C.M., Luu P.L., Peters T., Khoury A., Qu W., Zotenko E., Stirzaker C, & Clark S.J. (2019). Replication timing and epigenome remodelling are associated with the nature of chromosomal rearrangements in cancer. Nature Communications, 10, 416.
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10

ChIP-seq Data Processing and Analysis

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ChIP-seq libraries were prepared using the TruSeq ChIP Library Prep kit (Illumina) and subjected to paired-end sequencing. Quality control and pre-processing of raw data were processed using the Fastp software (version 0.20.1). Reads were mapped to the hg19 genome (ftp://ftp.ccb.jhu.edu/pub/data/bowtie2_indexes/hg19.zip) with Bowtie 2 44 (link) and duplicates were removed using the Picard v.2.20 tool 45 (link). MACS14 (version 1.4.2) was used for peak calling (q value ≤ 1e-04) 46 . The Chipseeker 47 (link) package in R statistical environment (R x64 3.6.3) was used to annotate the genomic region of the peaks. The motif enrichment analysis was performed using homer HOMER v4.11.1 (http://homer.ucsd.edu/homer/motif/).
Cui G., Gao Z., Chang S., Narwade N., Chen Y., Poudel B., Lei K.M., Zhang W., Li G., Poon T.C, & Cheung E. (2022). TRIM37 Augments AP-2γ Transcriptional Activity and Cellular Localization via K63-linked Ubiquitination to Drive Breast Cancer Progression. International Journal of Biological Sciences, 18(11), 4316-4328.
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