EXPERIMENT_TITLE: GSM3523749: Hi-C_WS-sgKAT7-rep2; Homo sapiens; Hi-C
EXPERIMENT_DESIGN_LIBRARY_DESCRIPTOR_LIBRARY_CONSTRUCTION_PROTOC: Genomic DNA extraction protocol not provided. Cells were harvested and DNA was extracted 28, 42 days post-infection (day28, day42), as well as at the end of the experiment (day56) using the Qiagen DNeasy Blood and Tissue Kit to evaluate library representation at middle and final time points. And the initial point are represented by plasmid sequencing. PCR on gDNA was performed using PrimeSTAR HS DNA Polymerase following manufacturer's instructions (Takara). A two-step PCR method was used to amplify inserted sgRNA sequences and append molecular barcodes and Illumina adapter sequences. In the first PCR, primers were designed to target the flanking sgRNA sequences with a biotin in forward primer. Reaction conditions were as follows: 98 °C denature for 2 minutes followed by 14cycles of 98 °C for 10 seconds, 58.0 °C anneal for 30 seconds, 72 °C extension for 40 seconds, and a final single extension step at 72 °C for 5 minutes. Multiple reactions per library were performed to ensure adequate input library coverage, then combined and purified by C1 beads (Invitrogen) and used as a template for the second PCR. The second PCR primers added the Illumina Hi-Seq2500 adapter sequences and barcode sequences. The reaction conditions were the same as the first PCR except only 12 cycles were used.
STUDY_DESCRIPTOR_STUDY_ABSTRACT: Understanding the genetic and epigenetic bases of cellular senescence is instrumental to aging intervention. We performed genome-wide CRISPR/Cas9-based screens in two human mesenchymal precursor cell (hMPC) models of progeroid syndromes and identified hundreds of genes whose deficiency alleviated cellular senescence. Among them, KAT7, a histone acetyltransferase, ranked as a top hit in both models. Inactivation of KAT7 decreased H3 lysine 14 acetylation (H3K14ac), repressed p15INK4b transcription, and rejuvenated both physiologically and pathologically aged cells. Moreover, lentiviral vectors encoding Cas9/sg-Kat7 alleviated liver senescence and extended healthspan and lifespan of mice. Our findings demonstrate that CRISPR/Cas9-based genetic screening is a robust method for systematically uncovering unknown senescence genes, of which KAT7 may represent a new therapeutic target for aging intervention. Overall design: CRISPR screen, RNA-seq, ChIP-seq and Hi-C
SAMPLE_TITLE: Hi-C_WS-sgKAT7-rep2
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Pool_Member_@attributes_sample_title: Hi-C_WS-sgKAT7-rep2
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