Mouse SETDB1 iKO-ES cells were treated with Tam for 3 d. Cells were then treated with fresh culture medium containing 1% formaldehyde for 10 min and washed twice with ice-cold PBS containing protease inhibitors. Cell pellets were resuspended in SDS lysis buffer also containing protease inhibitors (200 µL lysis buffer for every 1 × 106 cells) and incubated on ice for 10 min. Cell lysate was sonicated (15 W for 10 sec, six times) to shear DNA to lengths between 100 and 500 bp. Subsequently, ChIP was performed according to the ChIP assay kit (Millipore17-295) instructions using antibodies against SETDB1 (Santa Cruz, no. 66884), EZH2 (Millipore, no. 17-662), H3K27me3 (Millipore, no. 07-449), and H3K9me3 (Active Motif, no. 39161). Eluted DNA was used for PCR, qPCR, or deep sequencing. For ChIP-qPCR analysis, the relative binding level of each gene was normalized against input. Primer sequences are listed in Supplemental Table S2. For ChIP-seq libraries, 10 ng of input chromatin DNA or ChIP DNA was processed using the ChIP-seq sample prep kit (Illumina). Gel-purified ChIP-seq library DNA was further purified by phenol-chloroform extraction and ethanol precipitation and was processed for cluster generation, 15-cycle sequencing, and sequence analysis using Illumina HiSeq. The summary of generated ChIP-seq data sets is listed in Supplemental Table S3.
Chip seq sample prep kit
Manufactured by Illumina
Sourced in United Kingdom
The ChIP-Seq Sample Prep Kit is a laboratory equipment product designed for the preparation of samples for chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. The kit provides the necessary reagents and protocols to perform the essential steps in the ChIP-Seq workflow, including chromatin shearing, immunoprecipitation, and library preparation.
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Lab products found in correlation
Hiseq 2500, by Illumina (3 mentions)
Genome analyzer iix, by Illumina (2 mentions)
Gaiix, by Illumina (2 mentions)
Chip assay kit, by Merck Group (1 mentions)
H3k27me3, by Merck Group (1 mentions)
Setdb1, by Santa Cruz Biotechnology (1 mentions)
H3k9me3, by Active Motif (1 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (1 mentions)
Genome analysis pipeline, by Illumina (1 mentions)
Protein g magnetic dynabeads, by Thermo Fisher Scientific (1 mentions)
Anti znf322 antibody, by Santa Cruz Biotechnology (1 mentions)
Ribo zero, by Illumina (1 mentions)
Rneasy kit, by Promega (1 mentions)
Bioruptor, by Diagenode (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Chip it express kit, by Active Motif (1 mentions)
C ebpβ, by Santa Cruz Biotechnology (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
Gel extraction kit, by Qiagen (1 mentions)
Hpych4iii, by New England Biolabs (1 mentions)
24 protocols using chip seq sample prep kit
1
ChIP-seq Analysis of Setdb1 in Mouse ES Cells
2
ChIP-seq protocol for hippocampal chromatin
3
ChIP-seq Sample Preparation and Analysis
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ChIP-seq sample preparation and computational analysis of ChIP-seq data were performed as described previously with minor modifications51 (link). Library construction: the libraries were constructed following Illumina ChIP-seq Sample prep kit. Briefly, ChIP DNA was end-blunted and added with an ‘A’ base so the adaptors from Illumina with a ‘T’ can ligate on the ends. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR. Primary analysis of ChIP-Seq datasets: the image analysis and base calling were performed by using Illumina’s Genome Analysis pipeline. The sequencing reads were aligned to the human genome UCSC build hg18 by using Bowtie2 alignment program. Only uniquely aligned reads were kept. The aligned reads were used for peak finding with HOMER (http://biowhat.ucsd.edu/homer ). Of note, promoter (TSS)-associated peaks were defined as those with peak center falling between 100 bp downstream of and 5,000 bp upstream of TSS region. Similarly, enriched motifs in identified peaks were searched using HOMER. YY1 ChIP-seq data were deposited in the Gene Expression Omnibus database under accession GSE69759.
4
CHART-seq Protocol for Genomic Analysis
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CHART was carried out as described [20 (link)]. CHART capture oligonucleotides were designed according to instructions in [20 (link)]. The pulldown oligonucleotides consisted of OLIGO SEQ-3′ end-TEG and were ordered from IDT. Sequencing libraries were constructed by the Yale Center for Genomic Analysis (YCGA) and Yale Stem Cell Genomics Core using Illumina CHIP-Seq Sample Prep Kit. Sequencing was performed on Illumina HISeq 2500 or 2000 instruments. Each CHART time point was prepared in two biological replicates. CHART-seq data were deposited in the Gene Expression Omnibus (GEO) under the accession number GSE121268.
5
Chromatin Immunoprecipitation for ZNF322
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Chromatin was prepared using E14 cells as previously described [55] (link). Sonicated chromatin was incubated with Protein G magnetic Dynabeads (Invitrogen) coated with 40 µl of anti-ZNF322 antibody (sc-102205, Santa Cruz) overnight. The beads were then washed and Elution buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) was added to the beads and incubated for 45 min at 68°C with agitating at 1400 rpm. The eluent was decrosslinked by pronase. Lastly, DNA was precipitated and dissolved in nuclease free water for real-time PCR.
For ChIP-seq, ChIP DNA library was prepared by utilizing the ChIP-seq Sample Prep Kit (Illumina). Sequencing was then performed using the Genome Analyzer IIx (Illumina) and reads were mapped to the M. musculus genome assembly mm9.
For ChIP-seq, ChIP DNA library was prepared by utilizing the ChIP-seq Sample Prep Kit (Illumina). Sequencing was then performed using the Genome Analyzer IIx (Illumina) and reads were mapped to the M. musculus genome assembly mm9.
6
ChIP-Seq and RNA-Seq in T47D Cells
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For chromatin immunoprecipitation-sequencing, a total of 5 × 107 T47D cells were used per ChIP assay according to a previously described protocol35 (link). Briefly, cells were crosslinked with 1% paraformaldehyde for 10 min at room temperature, quenched with glycine, and fixed chromatin was sonicated and immunoprecipitated with specific antibodies. Libraries were prepared with Illumina’s ChIP-Seq sample prep kit for next-generation sequencing.
For RNA sequencing, T47D cells were transfected with control siRNA, TRPS1 siRNA, or CHD4 shRNA. Seventy-two hours later, total RNA was extracted with the RNeasy Kit (Promega). Total RNA was depleted of rRNA using Ribozero (Illumina), followed by library preparation and next-generation sequencing. The Gene Expression Omnibus (GEO) accession number for the ChIP-seq and expression data reported in this paper is GSE114213.
For RNA sequencing, T47D cells were transfected with control siRNA, TRPS1 siRNA, or CHD4 shRNA. Seventy-two hours later, total RNA was extracted with the RNeasy Kit (Promega). Total RNA was depleted of rRNA using Ribozero (Illumina), followed by library preparation and next-generation sequencing. The Gene Expression Omnibus (GEO) accession number for the ChIP-seq and expression data reported in this paper is GSE114213.
7
ChIP-seq Analysis of AFF1 Binding
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In total, 5 × 107 A549 cells were used per ChIP or ChIP-seq assay. Cells were cross-linked in phosphate buffered saline containing 1% formaldehyde to the cell culture media at room temperature for 10 min, and cross-linking was quenched by glycine. Fixed chromatin was sonicated into 200–800 base pair fragments (Bioruptor, Diagenode) in chromatin immunoprecipitation (ChIP) lysis buffer 10 mM tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, and 0.5% N-lauroylsarcosine supplemented with protease inhibitor cocktail (Sigma). Chromatin extracts were incubated with AFF1 antibody and protein A agarose beads at 4 °C overnight. Immunoprecipitates were washed with radio immunoprecipitation assay buffer (50 mM Hepes-KOH [pKa 7.55], 500 mM LiCl, 1 mM EDTA, 1.0% NP-40, and 0.7% Na-deoxycholate) for five times and TE once. After the final wash, DNA was eluted and reverse cross-linked at 65 °C. DNA was then purified and used as a template for qPCR. For ChIP-seq, libraries were prepared with Illumina's ChIP seq sample prep kit.
8
ChIP-Seq Protocol for Genome-wide Profiling
9
Chromatin Immunoprecipitation Sequencing
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HUVECs were stimulated in the presence or absence of 100 μM I942 or F/R for 48 h at 37 °C in 5% (v/v) CO2. Cells were then fixed and chromatin was extracted and sheared using the enzymatic “CHIP-IT Express Kit” (Active Motif) according to the manufacturer’s instructions. Sheared chromatin from I942-treated, F/R-treated and non-treated cells was then immunoprecipitated at 4 °C, overnight with 4 μg of either C/EBPβ or c-Jun ChIP-grade antibodies (Santa Cruz). DNA fragments were eluted from immunoprecipitated chromatin and used to prepare a ChIP-SEQ DNA library for sequencing using the “ChIP-SEQ Sample Prep Kit” from Illumina, according to the manufacturer’s protocols. Briefly, the first step in library preparation was to convert any overhangs in the ChIP’d DNA into phosphorylated blunt ends. The 3′ ends were then adenylated and adaptors ligated onto the ends of the fragments. The library was then size selected on an agarose gel and eventually enriched by PCR. The enriched library samples were then loaded onto a flow cell at a concentration of 12 pM and cluster formation was done on an Illumina Cluster station. Samples were then sequenced on an Illumina GA IIX giving 76 bp reads.
10
Random-Primed PCR for Bisulfite-Converted DNA
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DNA is single-stranded following bisulfite conversion. To make DNA double-stranded and generate enough material for constructing a sequencing library, we performed a random-primed PCR amplification. Bisulfite converted DNA was subjected to a round of linear amplification using an adapter combined with a random 9-nucleotide (nt) primer [GTTTCCCAGTCACGGTC(N)9], purified, and PCR-amplified using primers against the adapter sequences. The PCR reaction was purified with MinElute PCR Purification Kit (Qiagen) and digested with HpyCH4III (NEB) to remove the adapter sequences. The digest was resolved on a 2% agarose gel; the smear in the range of 100–500 bp was excised, purified with the Gel Extraction Kit (Qiagen), and used for library construction with the ChIP-Seq Sample Prep Kit (Illumina) according to the manufacturer’s instructions. The library was sequenced on an Illumina GAII to collect paired-end reads of 36 nt each.
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