Computational protocol: Non B Form DNA Is Enriched at Centromeres

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Protocol publication

[…] CUT&RUN was performed as described (, ). In brief, K562 or Cos-7 cells were gently washed twice in room temperature Wash buffer [20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM spermidine and a Roche complete EDTA-free tablet (Sigma-Aldrich) per 50 ml], with 3 min centrifugation at 600×g, mixed with activated Concanavalin A-coated magnetic beads (Bangs Laboratories) and rotated 5–10 min. Beads were captured by placing on a magnet stand, decanted and resuspended in Antibody buffer (2 mM EDTA and antibody at 1:100 in Dig-wash [Wash buffer supplemented with 0.05% digitonin (Calbiochem)]). Antibodies used were CENP-A (Abcam ab13939, mouse monoclonal), CENP-B (Abcam ab25734), Histone H3K27me3 (Cell Signaling Technologies 9733), and IgG (either Antibodies Online ABIN102961 guinea pig anti-rabbit, or GeneTex GTX105137, rabbit anti-human mitochondrial RNA polymerase). After overnight binding at 4 °C with rotation, beads were captured and washed once or twice in Dig-wash. For the CENP-A samples, beads were resuspended in secondary antibody (Abcam Ab46540, Rabbit anti-mouse) in Dig-wash and incubated 1 h at 4 °C and washed once in Dig-wash. Beads were resuspended in Protein A-MNase (Batch #5 360 µg/ml) 1:500 in Dig-wash, incubated 1 h at 4 °C, washed twice in Dig-wash and resuspended in 100 µl Dig-wash. Tubes were placed at 0 °C, mixed with 2 µl 100 mM CaCl2, incubated at 0 °C for 30 min, and reactions were stopped by addition of 100 µl 340 mM NaCl, 20 mM EDTA pH8, 4 mM EGTA, 0.05% digitonin, 50 µg/ml glycogen, and 200 pg mono-nucleosomal S. cerevisiae (spike-in) DNA. Samples were incubated 10 min at 37 °C and centrifuged 5 min 16,000×g at 4 °C and the supernatant was treated with 25 µg/ml RNAse A (Thermo) 10 min at 37 °C. After phenol–chloroform–isoamyl alcohol and chloroform extraction, DNA was precipitated by addition of 2.5 vol 100% ethanol, chilled on ice, centrifuged 10 min at 4 °C at 16,000×g and the pellets were rinsed in 100% ethanol and air-dried. Pellets were resuspended in 1 mM Tris pH8 0.1 mM EDTA and used for standard Illumina library preparation. Paired-end PE25x25 sequencing was performed by the Fred Hutch Shared Genomics Resource. Data have been deposited in GEO (GSE102111). [...] CENP-A and CENP-B CUT&RUN data were aligned to human alphoid BAC reference sequences described previously () or to an α-satellite-containing Chlorocebus aethiops BAC sequence (GenBank accession AC239401.3). Read length histograms were generated by counting the Bowtie2-reported aligned paired-end fragment lengths and autocorrelation of the resulting read length distributions was performed using Numpy. H3K27me3 CUT&RUN data were aligned to repeat-masked versions of the hg38 and Chlorocebus sabaeus assemblies as described above. [...] Our published CENP-A ChIP-seq 100×100-bp Illumina data were subjected to the same adapter trimming and quality filtering steps described above prior to merging pairs using SeqPrep as described previously (). Merged pairs were aligned to alphoid reference sequences using Bowtie2 using the single-end mapping parameters described above. Data from ChIP-seq of S. cerevisiae Cse4 (), S. pombe Cnp1 (), and CENP-A in human neocentromere cell lines () were aligned using the paired-end mapping parameters described above. [...] Tomtom v4.12.0 () with default parameters was used to determine similarity of yeast CDEI-derived motifs to a library of consensus sites for 203 yeast transcription factors () and to a Reb1 motif defined using high-resolution ChIP-seq (). […]

Pipeline specifications

Software tools SeqPrep, Bowtie2, Tomtom
Application ChIP-seq analysis
Organisms Homo sapiens, Mus musculus