|Public on May 01, 2023
|cell line: HEK293
|Cell were harvested at maximum 90% confluency after transfection. Cell were transfected at 60-70% confluency in 10cm dish with 6ug of plasmid DNA, 5ul Lipofectamine 2000 with Optimem. Cells were collected 3days after transfection.
|HEK293 cells were grown in DMEM supplemented with 10% FBS and 1X penicillin-Streptomycin-Antimycotic. These were grown at 37oC and 5% carbon dioxide
|Genomic DNA was harvested by GenElute Mammalian Genomic DNA miniprep kit (Merck)
DRIP DNA (2 ng) was used to generate DRIP-0seq libraries with NEBNext Ultra II DNA library prep kit (New England Biolabs) according to manufactureres instructions. Samples were multiplexed and sequenced on the Illumina NextSeq 500 (Illumina) using the single end protocol with 75 read length (high ouotput kit).
|Illumina NextSeq 500
|library strategy: DRIP-Seq
Samples were multiplexed and sequenced on the Illumina NextSeq 500 using the stranded single end protocol with a read length of 75.
Raw reads were adaptor trimmed and filtered for short sequences using cutadapt v1.8.1 3
The resulting FASTQ files were analysed and quality checked using the FastQC program
The filtered reads were mapped against the human reference genome (Hg19) using the BWA alignment algorithm 4 (version 0.7.9a-r786 with default parameters)
Enriched regions of the genome were identified using the MACS2 peak caller 5 (version 126.96.36.19960309) using default parameters and reporting only peakswith a FDR cut-off (-q) < 0.05
Supplementary_files_format_and_content: MACS2 peak files
|Jan 31, 2019
|Last update date
|May 01, 2023
|College of Medicine and Public Health
|Circular RNAs in Cancer Laboratory
|Sturt Road, Bedford Park
|Circular RNAs drive oncogenic chromosomal translocations through R-loop-mediated genome instability [DRIP-seq]
|Circular RNAs drive oncogenic chromosomal translocations through R-loop-mediated genome instability