|Public on May 01, 2023
|K562 MIG EV 2
|cell line: K562
cell type: CML
genotype: cell line
treatment: Transgenic line
|Cells stably transduced with lentivirus were selected on the basis of GFP expression encoded on the pMIG plasmid by flow cytometry. Cell populations with GFP expression >99% were used for experiments.
Cells were xenografted into NSG mice (6-8 per group) in a single-blinded experiment. Mouse welfare was tracked and weekly bleeds were performed to look at prpoportion of human GFP cells in the circulation. Mice were euthanised as a result of tumour cell burden and cells were collected, and following RBC lysis, were sorted by flow cytometry for GFP expression. Cell lines were made and frozen down within 72hrs post-collection.
|K562 and HL-60 cells stably expressing transgenic constructs were cultivated in RPMI1640 medium with 10% FBS at 37oC with 5% carbon dioxide
|RNA was extracted with TRIzol and purified with Zymo RNA purfiication kit with on-column DNaseI digestion.
NEBNext UltraII Directional RNA sequencing kit
|Illumina HiSeq 2500
|Raw reads were adaptor trimmed and filtered for short sequences using cutadapt v1.8.1 (Martin, 2011), setting minimum-length option to 18, error-rate 0.2 and overlap 5.
The resulting FASTQ files for total RNAseq respectively were analysed and quality checked using the FastQC program (http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Reads were mapped against the human reference genome (hg19) using the STAR spliced alignment algorithm (Dobin et al., 2013) (version 2.5.3a with default parameters and --chimSegmentMin 20).
The resulting STAR produced Chimeric.out.junction file for each sample was parsed and annotated for circRNA prediction and backsplice abundance using CIRCexplorer2 (Zhang et al., 2016).
Alignments were visualised and interrogated using the Integrative Genomics Viewer v2.3.80 (Thorvaldsdóttir et al., 2013).
|Oct 30, 2022
|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
|Circular RNAs drive oncogenic chromosomal translocations through R-loop-mediated genome instability [RNA-seq 2]