Project description:We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences.
Project description:We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Chromatin pull down of hetR mutant strain UHM103 carrying pAM4375, which expresses HetR-6xHis, and a wild-type control. One dataset was collected.
Project description:RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ~10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide 1.sgRNA1-6 binding sites were identified with ChipSeq by using HA antibody (there are 2 replicates for sgRNA1-3, one sample for sgRNA4-6,one control without sgRNA) 2.PCR products which amplifies " off-target genomic sites" were deep sequenced in the presence of WT Cas9+sgRNA or WT Cas9 alone( unique adaptor was used for each sgRNA and mixed for multiplex run)
Project description:Single cell full transcriptome sequencing of CD19 CAR T-cell infusion products used for standard of care treatment for relapsed/refractory large B-cell lymphoma.
