![]() In four GBM tumours, we characterised one probable endonuclease-independent L1 insertion, two L1-associated rearrangements and one likely Alu- Alu recombination event adjacent to an L1. Here, using retrotransposon capture sequencing (RC-seq), we surveyed L1 mutations in 14 tumours classified as glioblastoma multiforme (GBM) or as a lower grade glioma. Recent reports suggest that L1 is mobile in epithelial tumours and neural cells but, paradoxically, not in brain cancers. Notably, cancer cells can support unusual L1 retrotransposition and L1-associated sequence rearrangement mechanisms following DNA damage. GSM6448279_KO52_EV_ATAC_rep1_treat_-1 (L1) retrotransposons are a notable endogenous source of mutagenesis in mammals. Identification and interrogation of the gene regulatory network of CEBPA double mutant Acute Myeloid Leukaemia ![]() Institute for Cancer and Genomic Sciences Supplementary files format and content: bedGraph files of read pileups generated by MACS2 Open chromatin regions (peaks) were identified using MACS2 and bedGraph files were generated by including the options -B -trackline PCR duplicated reads were removed using the MarkDuplicates funtion Picard tools Reads were then mapped to the human genome version hg38 using Bowtie v2.2.3 Sequencing adapters and low-quality reads and were removed using Trimmomatic KO52 were cultured in Alpha MEM (Lonza) supplemented with 10% FCS (Gibco), 5% Pen-Strep, 5% L-Glu.ĪTAC libraries were generated following the Omni-ATAC protocol from Corces et al, 2017 (PMID:28846090) Dominant negative peptides expression was induced by adding 2 μg/mL of doxycycline to culture medium. Despite several attempts, it was not possible to expand KO52 single cell clones with either vector and the bulk population was used for downstream experiments. Single clones were then isolated from the bulk Kasumi-1 population, expanded and tested for leakiness and induction of dnC/EBP measuring both GFP and FLAG expression through both FACS and RT-qPCR. Few days after infection, cells were cultured in medium supplemented with puromycin 1.5 ug/mL and 2ug/mL for 5 days, allowing the selection of cells positive for pCW57.1-dnC/EBP. After infection cells were incubated at 37 ☌ for 12 hours and then resuspended in fresh media. To carry out the spin infection, 1 x 10^6 Kasumi-1 or KO52 cells were plated in 17 6-well plates with the viral supernatant and 8 ug/mL of polybrene, and centrifuged for 1h 30m at 1500 x g at 32☌. Concentrated viral particles were filtered through 0.45 um filter and stored at -80☌ or immediately used to infect recipient cells. Viral supernatant was harvested at 24, 36, 48, 60 and 72h and concentrated with Centricon Plus- 70 columns (Merk-Millipore) using manufacturer’s instructions. ![]() 12 μg of pCW57.1-dnC/EBP or empty vector were transfected with 0.6 ug of REV, 0.6 ug of GAG/POL, 1.2 ug of VSV-G and 0.6 ug of TAT packaging plasmids. Cells were cultured in DMEM supplemented with 10% foetal calf serum (FCS), 5% Penicillin-Streptomycin (Pen-Strep), 5% L-Glutamine (L-Glu) and 5% Sodium Pyruvate and seeded to achieve about 30-40% confluency at the time of transfection. Lentiviral particles containing pCW57.1-dnC/EBP / dnFOS or the empty vector were generated in HEK293T cells using calcium-phosphate transfection. The pCW57.1 containing the dnFOS insert was generated by Dr Sandeep Potluri (University of Birmingham) following the same protocol. The dnC/EBP insert was then transferred into the Tet-on pCW57.1 backbone (Addgene plasmid #41393) by using the Gateway Gene Cloning System. dnC/EBP cDNA was PCR amplified (with Phusion DNA polymerase (New England Biolabs) using primers containing SalII and NotI restriction enzymes overhangs and cloned into pENTR-IRES-GFP (a modification of the Invitrogen pENTR plasmid constructed by Benjamin Edginton-White, University of Birmingham, UK) digested with 30 units of SalI-HF (New England Biolabs), 30 units of NotI-HF (New England Biolabs). The CMV500 A- C/EBP plasmid containing the dnC/EBP insert was a gift from Charles Vinson, National Cancer Institute, Bethesda, USA 1. The doxycycline inducible pCW57.1-dnC/EBP plasmid was generated using Gateway Gene Cloning (Thermo Fisher Scientific), according to manufacturer’s instructions. GEO help: Mouse over screen elements for information.
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