edCas9 WCE minus r2
Saccharomyces cerevisiae cultures with pPrA-dCas9 + gRNA-ARS305 were grown to mid-log phase in synthetic yeast media (minus tryptophan) with 2% w/v raffinose and subjected to 1% formaldehyde cross-linking for 5 minutes. Cells were collected by centrifugation. Lysate from 1.5 g of cells (~5x1010 cells) was re-suspended in cold purification buffer. PrA-tagged dCas9/gRNA complex and associated chromatin were affinity purified on 5 mg of M270 Dynabeads conjugated to rabbit IgG per gram of cells. IgG-coated beads were incubated with lysate for 3 hours at 4C with gentle rotation. Beads were collected with magnets and washed once in purification buffer, once with purification buffer with 1 M urea / 1 M NaCl, once in purification buffer with 4 M urea/250 mM LiCl, once in purification buffer with 250 mM LiCl, and once more in purification buffer. DNA was eluted from the beads in 100 l of 25 mM Tris pH 8, 2 mM EDTA, 1% SDS by heating for 20 minutes at 95C, followed by incubation at 65C overnight (~16 hours). The eluate was then treated with 20 g RNase A for 30 minutes, then 20 g proteinase K for 30 minutes at 37C. The DNA was subsequently purified on silica columns using a Promega SV Gel and PCR cleanup kit. These samples were then used for sequencing sample preparation with the TruSeq ChIP Library Preparation Kit from Illumina. Raw reads obtained from the CRISPR-ChAP libraries were first examined for quality using the FastQC software. Then the possible remaining adaptors and raw reads were quality trimmed using Trimmomatic with default setting to obtain high quality reads. The high-quality reads were mapped to the reference genome (Saccharomyces cerevisiae S288C, GCF_000146045.2), retrieved from NCBI database along with the corresponding annotation files, using BWA-MEM ollowed by Stampy aligner to improve mapping accuracy.