Thruplex dna seq kit

ChIP was carried out as described (Jehle et al., 2014 (link)), using anti-AR antibody (N-20; Santa-Cruz Biotechnology). Following primers were utilized for directed ChIP qPCR. KLK3 (AREIII) primers were previously published (Jehle et al., 2014 (link)).
TMPRSS2 Fwd: 5’-GCTCACACAGGATCAGAGCA-3’
TMPRSS2 Rev: 5’-TGCTCGTTAGTGGCACATTC-3’
NKX3.1 Fwd: 5’-TTTGGGCCACCCTGTAAATA-3’
NKX3.1 Rev: 5’-GGGTGGGAGGAGATGAAAAT-3’
ChIP-seq libraries were generated using the ThruPLEX DNA-seq kit (Rubicon Genomics, Ann Arbor, MI) and were sequenced on the Illumina NextSeq 500 platform at the Molecular Biology Core Facility (Dana-Farber Cancer Institute). ChIP-seq data was processed using ChiLin2 (Qin et al., 2016 (link)). All ChIP-seq data have been deposited at the GEO depository under accession number GSE89939.

ChIPed DNA was tested for quality with BioAnalyzer (Agilent) before library preparation. Libraries were constructed with ThruPLEX^® DNA-seq Kit (Rubicon Genomics) following the manufacturer’s instructions. High-throughput sequencing was performed with the Illumina HiSeq 2000 Sequencing System. ChIP-seq reads were aligned to the mouse genome mm10 using Bowtie2 (v2.2.6) with default settings. For histone modifications, the resulting BAM files were filtered to remove the reads that have the same sequence, and converted to reads coverage files using the BamCoverage in DeepTools (v2.5.0). Reads coverage files were normalized as RPKM (Reads Per Kilobase of transcript per Million mapped reads) and visualized in IGV (v2.3). Heatmap graphs were generated with DeepTools (v2.5.0). For transcription factors, filtered BAM files were used for peak calling and differential peak analysis using MACS2 (v2.1.1). Differential peaks were annotated with PAVIS (https://manticore.niehs.nih.gov/pavis2/). GO analysis of differential peaks was performed with DAVID (https://david.ncifcrf.gov). Motif analysis was performed by HOMER software.

ChIP DNA library construction was performed by using ThruPlex DNA-seq Kit (Rubicon Genomics, Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, 10 μl of ChIP DNA or 10 ng of input DNA was used for library construction and PCR amplification was performed to obtain the final library. AMPure XP beads were used for library purification and purified libraries were analyzed in Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA) to verify fragment size after amplification, library size and concentration before clustering. A total of 10pM of the library was then used for single-end sequencing on the HiSeq 2500 at the Genomic and RNA Profiling Core in Baylor College of Medicine. Sequences (51-nt reads) were aligned to the mouse genome (mm9) using the BWA algorithm. Aligns were extended in silico at their 3’-ends to a length of 150bp and assigned to 32-nt bins along the genome. The resulting histograms were stored as Binary Analysis Results (BAR) files. Peak locations were determined using the Model-based Analysis of ChIP-seq Algorithm (MACS) with a p-value cutoff of 1E-8.

ChIP-Seq was carried out using the method previously described using a biological duplicate (58 (link)). Briefly, 5 × 10⁶ mesenchymal chondrosarcoma cells per immunoprecipitation were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Chromatin was sheared in SDS lysis buffer containing 1% SDS, 10 mM EDTA, and 50 mM Tris, pH 8.0, to an average size of 400–500 bp using a Covaris S220 sonicator for 15 minutes. ChIP was carried out with 5 μg anti-FLAG (Sigma-Aldrich), anti-histone H3K27ac (Active Motif), anti-histone H3K27Me3 (Abcam), anti-histone H3K4Me3 (Abcam), anti-Runx2 (Cell Signaling), or anti-Runx3 (Cell Signaling) antibodies. The antibody-bound protein/DNA complexes were immunoprecipitated using ChIP grade protein G magnetic beads (Cell Signaling).
Immunoprecipitated DNA was then purified and subjected to secondary sonication to an average size of 150–350 bp. Libraries were prepared according to instructions accompanying the ThruPLEX DNA-Seq kit (Rubicon Genomics). The ChIP DNA was end modified and adapters were ligated. DNA was PCR amplified with Illumina primers, and Illumina-compatible indexes were added. The library fragments of approximately 300-500 bp were band-isolated from an agarose gel. The purified DNA was sequenced on an Illumina MiSeq next-generation sequencer following the manufacturer protocols.