Project description:A genome-wide screening identified effector genes of chromatin accessibility. To investigate the functional consequences of the loss of each effector gene, we conducted ATAC-seq analysis with eHAP cells knocked out for each effector gene.
Project description:A genome-wide screening identified effector genes of chromatin accessibility. To investigate the functional consequences of the loss of each effector gene, we conducted Mnase-seq with eHAP cells knocked out for TFDP1.
Project description:Cas9 expressing eHAP cells were transfected with sgRNA against TFDP1. 5 days after the transfection, polyA RNA was analyzed by RNA-seq
Project description:eHap CRISPR-Cas9 mutagenized cells with no selection serves as a control. RPS6p antibody staining on the mutagenized eHap cells was used in a flow-sorting approach to isolate RPS6p high and low populations respectively. The genomic DNA from these different samples was isolated, subjected to PCR to amplify the genomically-encoded guide RNA sequences, the product(s) of which was then sequenced on a Hi-Seq X Ten instrument by Novogene.
Project description:eHAP or U87MG cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million eHAP or U87MG cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the Brunello library at MOI 0.3. Cells were then selected with puromycin, expanded to 3 billion cells, and then pooled together and cryofrozen in aliquots. One hundred million cells were thawed constituting over 1000× genome coverage worth of mutagenized library. Twenty T175 flasks were used for the U87MG-based screen, while twelve T175 flasks were used for the eHAP-based screen. The cells were allowed to recover for 48 hours before infecting with ReoT3D at an MOI of 0.1. Obvious CPE was observed within 72 hours. eHAP-resistant colonies were harvested two weeks later, while U87MG-resistant colonies were harvested six weeks later. The uninfected reference used was the unselected starting population. The unselected and selected cells were both processed with QIAamp DNA columns to purify the gDNA. A first round of PCR was used to amplify the guide RNA sequences encoded in the gDNA, followed by a second round of PCR to add the barcodes/adapters for amplicon sequencing. 2% agarose gels and a QIAquick gel extraction kit were used to purify the amplicons. The amplicons were then subjected to next-generation sequencing on a HiSeq instrument lane (Illumina) via Novogene.
Project description:Interventions: experimental group :PD-1 Knockout Engineered T Cells
Primary outcome(s): Number of participants with Adverse Events and/or Dose Limiting Toxicities as a Measure of Safety and tolerability of dose of PD-1 Knockout T cells using Common Terminology Criteria for Adverse Events (CTCAE v4.0) in patients
Study Design: historical control