Project description:Molecular phenotyping quantifies expression of pathway reproter genes by next-generation sequencing of pre-defined amplicons. We applied the technology to quantify pathway-level impact of ASOs to PTECs in the presence and absence of exogeneous EGF.
Project description:We use targeted bisulfite PCR and next-generation 454 sequencing of multiple amplicons to analyze the association of cis-regulated allele-specific methylation (ASM) with multiple complex disease-associated variants in a population of 82 individuals. We detect ASM at four variants implicated in complex phenotypes such as ulcerative colitis and AIDS progression disease (rs10491434), Celiac disease (rs2762051), Crohn’s disease, IgA nephropathy and early-onset inflammatory bowel disease (rs713875) and height (rs6569648). 82 samples analysed
Project description:In order to clarify the mechanism to MYC (8q24) amplification as dmin or hsr, we finely investigated the organization of amplicons in seven tumor cell lines [four SCLC, one AML, and two Colon Carcinoma (CC)] by integrating next-generation sequencing (NGS), Fluorescent in situ hybridization (FISH), PCR, and array-based approaches.
Project description:Huh7.5.1 cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, Huh7.5.1 cells that constitutively express Cas9 were transduced with lentiGuide-Puro from the druggable genome library at MOI 0.3. Cells were then selected with puromycin, expanded, and then pooled together and cryofrozen in aliquots. Cells were thawed constituting over 1000× genome coverage worth of mutagenized library. The cells were infecting with DENV2_429557 and DENV-2_16681_Hap1-adapted at MOI of 0.1. Virus-resistant colonies were harvested. 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:HT29-DKO cells were stably transduced with lentiCas9-Blast (Addgene, #52962) and subsequently selected using Blasticidin. Then, 300 million HT29-DKO 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. The cells were infecting with PeV-A1 or PeV-A2 at an MOI of 0.1. Virus-resistant colonies were harvested. 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:Next Generation Sequencing in cancer: a feasibility study in France to assess sample circuit and to perform analyzes within a limited time.
