ChIP assays were performed as described above. Recovered DNA (~100 ng) was then used for library preparation using the TruSeq ChIP Sample Preparation protocol following Manufacter’s instructions (Illumina Technologies). Paired-end sequencing was then performed with the Illumina HiSeq 2000 instrument. More than 20 millions of single reads were obtained for RPC1 and RPB1 libraries and 10 millions of single reads for IgG library. The single-matched reads were mapped to an hybrid ovine genome (OAR v3.1) containing the BLV provirus sequence (GenBank: KT122858.1) at the L267 integration site (chr10:86299813) using bowtie2 software (-N 1 and -k 5 parameters). Finally, our results were visualized using IGV program. Reads spanning LTR-host and LTR-BLV boundaries were extracted from the alignment file using an in-house R script and ggplot2 was used to plot the ChIP-seq coverage.
Truseq chip sample preparation protocol
Manufactured by Illumina
The TruSeq ChIP Sample Preparation protocol is a laboratory workflow designed to prepare chromatin immunoprecipitation (ChIP) samples for sequencing on Illumina sequencing platforms. The protocol includes steps for chromatin fragmentation, immunoprecipitation, and library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Dynabeads, by Abcam (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab31419, by Abcam (1 mentions)
Ab81086, by Abcam (1 mentions)
Genome analyser iix, by Illumina (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
Hiscansq, by Illumina (1 mentions)
6 protocols using truseq chip sample preparation protocol
1
ChIP-seq Profiling of BLV Provirus
2
ChIP-seq Library Preparation and Analysis
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Libraries were prepared using a modified version of the Illumina TruSeq ChIP sample preparation protocol. See the the Supplemental Material for complete details. Each library (10 pM) was sequenced on an Illumina HiSeq 2000. The raw reads in ChIP-seq data sets were mapped to NCBI build 37 (University of California at Santa Cruz [UCSC] mm9) using Bowtie (version 0.12.8) (Langmead et al. 2009 (link)). Peaks were called using MACS (version 2.0.10) (Zhang et al. 2008 (link)) at a Q-value threshold of 0.05 using input as a control. The enriched regions from each biological replicate of samples were intersected with BEDTools (version 2.13.3) (Quinlan and Hall 2010 (link)) to form the final peak set for Gcn5 bioChIPs.
3
ChIP-qPCR and ChIP-seq Analysis of Transcription Factor Binding
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ChIP-qPCR and ChIP-seq were carried out as described previously [13 (link)]. For ChIP-qPCR for AP1, a JUN antibody (Abcam (ab31419)) was used and for histone acetylation a H3K27ac antibody (Abcam (ab4729)) was used. For ChIP-seq, 3x107 cells, 3 μg antibody (ETV1; Abcam (ab81086)) and 30 μl Dynabeads were used per experiment. Parallel control experiments were run with ChIP-qPCR using rabbit IgG; Millipore (12–370). Library preparation was performed using the TruSeq ChIP Sample Preparation Protocol (Illumina) and DNA libraries were sequenced using the HiSeq 2500 (Illumina).
Sequencing tags/reads from the ETV1 ChIP-seq experiment in OE33 cells were aligned to the NBCI Build hg19 of the human genome with Bowtie v2.2.3 [38 (link)]. Up to two mismatches were allowed. Only reads with a mapping quality >q30 were retained. Peak calling was performed on individual replicates and merged datasets with MACS v2.1.0 software [39 (link)] using default parameters. Data are deposited in ArrayExpress (Accession number: E-MTAB-5168)
Sequencing tags/reads from the ETV1 ChIP-seq experiment in OE33 cells were aligned to the NBCI Build hg19 of the human genome with Bowtie v2.2.3 [38 (link)]. Up to two mismatches were allowed. Only reads with a mapping quality >q30 were retained. Peak calling was performed on individual replicates and merged datasets with MACS v2.1.0 software [39 (link)] using default parameters. Data are deposited in ArrayExpress (Accession number: E-MTAB-5168)
4
Illumina Sequencing Library Preparation from SELEX
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The single-stranded DNA (ssDNA) obtained after each round of SELEX underwent library preparation for Illumina sequencing using the TruSeq ChIP sample preparation protocol. A total of nine DNA libraries comprised of paired-end indexed sequences were created by pooling the samples. The Illumina TruSeq ChIP sample preparation kit reagents were utilized for this process to facilitate cluster generation and subsequent DNA sequencing. The initial DNA input (50 µL of 200 pg/µL) underwent processes to blunt-end and phosphorylate the molecules. Additionally, a single “A” nucleotide was attached to the 3 ends of the fragments, preparing them for ligation to adapters featuring a single-base “T” overhang. These adapter sequences were introduced to the DNA ends to generate either indexed single-read or paired-end sequencing libraries. Following ligation, the resulting products were purified and accurately size-selected using agarose gel electrophoresis. The DNA fragments of the desired size were isolated and purified once more. Subsequently, a PCR amplification step was performed to enrich for fragments possessing adapters on both ends. The final product underwent quantification prior to the initiation of cluster generation.
5
ChIP-seq Analysis of FOXM1 Transcription Factor
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ChIP-qPCR and ChIP-seq were carried out as described previously [16 (link)]. For ChIP-seq, 3×107 cells, 3 μg antibody (FOXM1; Santa Cruz Biotechnology (sc- 502 X) or rabbit IgG; Millipore (12–370)) and 30 μl Dynabeads were used per experiment. Library preparation was performed using the TruSeq ChIP Sample Preparation Protocol (Illumina) and DNA libraries were sequenced using the Genome Analyser IIx (Illumina).
6
ChIP-seq Library Preparation and Sequencing
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Sequencing libraries were prepared according to the Illumina’s TruSeq ChIP Sample Preparation protocol. In brief, the enriched ChIP DNA was end-repaired and indexed adapters were ligated to the inserts. Purified ligation products were then amplified by PCR. Amplified libraries were prepared in the Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen, Debrecen, Hungary (Halász et al., 2017 (link)). The libraries were sequenced using 50 single-end reads with Illumina HiScan SQ (Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen); or with Illumina HiSeq 2500 (EMBL Genomics Core Facility, Heidelberg, Germany).
Raw reads were aligned to the S. cerevisiae reference genome (SacCer3; SGD) using the default parameters of Burrows-Wheeler Aligner algorithm (Li and Durbin, 2009 (link)) and 38–67% of the sequenced reads were retained after removing low mapping quality (MAPQ < 10) and PCR duplicate reads (Picard).
Raw reads were aligned to the S. cerevisiae reference genome (SacCer3; SGD) using the default parameters of Burrows-Wheeler Aligner algorithm (Li and Durbin, 2009 (link)) and 38–67% of the sequenced reads were retained after removing low mapping quality (MAPQ < 10) and PCR duplicate reads (Picard).
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