Project description:Inserting large DNA payloads (>5 kb) into specific genomic sites of mammalian cells remains challenging. We have merged the strengths of different classes of site-specific recombinases and combine these with CRISPR/Cas9-mediated homologous recombination to develop a strategy for targeted DNA integration of huge constructs (e.g. >170 kb) as well as stringent site-specific replacement of genomic fragments >50 kb in size in human induced pluripotent stem cells. In order to validate the genome integrity of the payloads integrated by STRAIGHT-IN (Serine and Tyrosine Recombinase Assisted Integration of Genes for High-Throughput INvestigation), we performed next generation sequencing (i.e. whole genome and targeted capture sequencing) on the resulting genetically modified cell lines. We have deposited here the raw data.
Project description:iMUT-seq profiles double-strand break (DSB) induced mutations at extremely high sensitive with single nucleotide resolution, allowing for the quantification of all mutation types, including chromosomal rearrangements, around endogenous DSBs. AID-DIvA cells are treated with or without 4-hydroxytamoxifen to induce DSBs, then with IAA to induce degradation of the DSB inducing AsiSI enzyme and are then incubated to allow the breaks to be repaired. The DSB loci are then PCR amplified from the genomic DNA and subjected to NGS to provide high depth mutation profiling. All samples are either untreated (m/0) or treated (p/4)with OHT and have three independent biological replicates, with the exception of ATRi which has 2 replicates and controlsi which has 6 replicates. This is because the siRNA were divided into two batches to create replicates 1-3 and 4-6 and each batch contained a controlsi sample for reference causing it to be in all 6 replicates.
Project description:RpfB is one of five resuscitation promoting factors in Mycobacterium tuberculosis. We previously published data indicating the 5' UTR of the gene contains a regulatory riboswitch with an unknown (at the time) ligand. We overexpressed the switch in Mycobacterium smegmatis (which does not contain the rpfB gene), reasoning that overexpression of the switch would s sequester the unknown ligand, giving rise to a phenotype that would help us identify it.
Project description:HT-29 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib resistant derivatives of these cell lines were established, respectively. Five million barcoded HT-29 cells were seeded into 15 cm cell culture dishes. When the cells reached confluency, two million cells per dish were seeded into four different 15 cm dishes (DMSO Control, Replica A, B, C) and two million cell pellets were stocked as initial cell population. Harvesting used medium through the experiment was performed at monthly intervals. Barcoded HT-29 cell line replicates A, B, and C were treated with 2XIC50 (199.6 nM) of dabrafenib concentration for the duration of 3 months.Barcoded data can be accessed via accession code E-MTAB-13018. Whole exome sequencing of dabrafenib-resistant A replicate and DMSO control cell lines were carried out.
Project description:HT-29 and HCT-116 cells were barcoded using the CloneTracker lentiviral barcode library and then dabrafenib and irinotecan resistant derivatives of these cell lines were established, respectively.10 million barcoded HT-29 and HCT-116 cells were seeded equally onto poly-HEMA coated 4xT75 flask (DMSO Control, Replica A, B, C for each drug). After seeding, cells were allowed to form spheroids and barcoded 3D-HT-29 spheroids were treated with dabrafenib at increasing doses starting from IC50/10 dose until IC50/2 dose with monthly doubling of the dosing (16 weeks), and barcoded 3D-HCT-116 cells were treated with irinotecan at increasing doses starting from IC50/4 dose until IC50 dose with weekly doubling of the dosing (4 weeks). Following the end points of treatment for each cell line, DNA was isolated from harvested cell lines and barcode sequencing and whole exome sequencing were carried out.
Project description:The mammalian genome has an abundance of transposable elements, but understanding their contribution to complex biological systems is poorly understood. Here, we report the CRISPR/Cas9 deletion of a retrotransposon (Lx9c11) in mice and its effect on the immune response to virus infection. The regulatory role for Lx9c11 was assessed by RNA-seq analysis of knockout and wild-type mice, treated with CVB4 virus. Long read nanopore RNA-seq of transcripts enriched from Slfn1 locus (mm10, chr11:83109157-83116657).
Project description:Whole exome sequencing was performed on set of 48 DNA samples obtained from 16 EGFR mutated NSCLC patients whose tumors progressed following EGFR-TKI treatment. The DNA samples included baseline biopsy, rebiopsy and blood from the same patient. By comparing the variants in rebiopsy tumors and baseline tumors we aim to understand the genomic alterations responsible for the development of EGFR-TKI resistance in NSCLC patients.
Project description:BANC-seq (Binding Affinities to Native Chromatin by sequencing) allows determination of absolute apparent binding affinites of transcription factors to native chromatin in a genome-wide manner. In this study, we establish the method and show that chromatin and DNA sequence define binding affinites of FOXA1, SP1, YY1 and MYC/MAX complex. To relate the identified binding affinities to the actual transcriptional levels of these transcription factors in the cells used in this study, and to confirm that the nuclear isolation protocol used in the study does not lead to loss of nuclear proteins, we have generated whole proteomes including proteomics standards for absolute quantification of proteins. In addition, to validate some of the findings of the study, we have performed PAQMAN (Protein-nucleic acid affinity quantification by MAss spectrometry in nuclear extracts), as well as DNA pulldowns followed by mass spec.
Project description:Transcription profiling of mouse FR4+Egr2+, FR4+Egr2- and FR4-Egr2- CD4+ T cells extracted from GFP-Egr2 knockin (Egr2 Kin) and FR4+Egr2-/-Egr3-/- and FR4-Egr2-/-Egr3-/- CD4+ T cells isolated from Egr2loxP/loxP hCD2-Cre Egr3-/- (Egr2/3 DKO) mice in the steady state or 21 days after infection with vaccinia virus.