The libraries were quantified using a Qubit and Agilent bioanalyzer, pooled and subjected to 150 bp single-end read sequencing with an Illumina NextSeq sequencer. Libraries were constructed from ChIP and input DNA by standard Illumina protocols, except that DNA in the range 150-350bp was gel-purified after PCR-amplification.
IP and input DNA were then purified by treatment with RNase A, proteinase K and phenol:chloroform extraction followed by ethanol precipitation. Bound complexes were eluted from the beads by heating at 65☌ with occasional vortexing and crosslinks then reversed in IP and input DNA by overnight incubation at 65☌. Beads were washed 6 times with RIPA buffer and 1 time with TE containing 50 mM NaCl. The resulting whole cell extract was cleared by centrifugation and then incubated overnight at 4☌ with 100 µl of Dynal Protein G magnetic beads that had been pre-incubated with 20 µl of anti-T-bet serum. Mean of input DNA and H3 coverage over Top15 resected AsiSI DSBs in control and Sth1 Snf2 depleted cells shows the extent of DNA and H3 signal loss at the. (F) Resection and histone eviction are severely impaired in Sth1 Snf2 depleted cells. Cells were sonicated on ice to solubilize and shear crosslinked DNA (24W for 10 x 30 second pulses using a Misonix Sonicator 3000). (E) Strand-specific H3 ChIP-seq read coverage for WT and Sth1 Snf2 depleted cells at Chr VII 384,688 bp. Cells were lysed with non-ionic detergent, nuclei washed and then lysed with ionic detergent. Cells were rinsed twice with 1xPBS and flash frozen in liquid nitrogen.
Library_strategy ChIP-Seq library_source GENOMIC library_selection ChIP library_construction_protocol Cells were crosslinked by the addition of one-tenth volume of fresh 11% formaldehyde solution for 20 minutes at room temperature before the reaction was quenched by addition of glycine.
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