Presumably, an interaction between cellular factor(s) and lentiviral CA affects virus uncoating, thus influencing downstream events such as nuclear import and integration [35,36,38]. contamination. The same cDNA preparations were utilized for amplification of the GAPDH mRNA. As a positive control 100?pg of plasmid DNA (pGR102; ) was used. Mock: mock transduced cells; NTC: no template control. Physique S4. Ex lover vivo transduction of hematopoietic lineage unfavorable (Lin?) cells derived from murine bone marrow. Bone marrow cells were enriched for the population of Lin? cells using the Lineage Cell Depletion kit (Miltenyi Biotech). The enriched cells were analysed by FACS using anti-CD117-PE antibody (Miltenyi Biotech)(upper panel). The cells were cultured overnight in the StemSpan medium either made up of or lacking a murine cytokine cocktail consisting of 100?ng/ml rmSCF, 100?ng/ml rmTPO, 100?ng/ml rmFlt3-L and 20?ng/ml IL3. Following the 16?h pre-incubation period, the cells (1 106 cells/ml) were transduced with MMTV-or HIV-1-based vectors (1.8 107 TU/ml). 24?h after transduction, the vector-containing supernatant was replaced with StemSpan medium with or without the murine cytokines. Five days after transduction the cytokine-containing medium was added to cells and they were cultured for 10?days. FACS analysis was used to detect the expression of EGFP in the Lin? cells. 1742-4690-11-34-S1.pdf (1.3M) GUID:?7257A597-F2AA-43DA-A62D-5D053ACE15F8 Additional file 2: Physique S5 Comparison of integration site distribution within genes, tumor associated genes, CpG islands and open chromatin regions. Nucleotide coordinates for the genomic features were obtained from the USCS KRAS G12C inhibitor 17 Genome Bioinformatics Table Browser using NCBI36/hg18 assembly of the human genome. For the tumor associated genes, The Malignancy Gene Atlas (TCGA) Gene Ranker and Tumor Associated Gene Database (NCKU Bioinformatics center) were used to obtain a list of genes. A Chi-square test was used to test differences between random dataset and vectors. Figure S6. Comparison of integration site distribution within or near (simple repeats: 1?kb; tRNA and miRNA: 10?kb) various genomic features. Nucleotide coordinates and statistical evaluation of differences was performed us explained above. The GC content in a 100?nt windows surrounding the integration sites was computed and the KRAS G12C inhibitor 17 frequency of integrations in the loci with GC content exceeding 41% (average GC content for the human genome) was plotted for vectors and random dataset. Physique S7. Gene density around integration sites. RefSeq genes found in the vicinity (1?Mb window) of integration sites were counted. The average and mean quantity of genes for vectors and random sites are shown. The middle plots show the frequency of integrations in the regions with a gene density of 0-10 KRAS G12C inhibitor 17 genes, 11-20 genes and >20 genes within the 1?Mb windows. A Chi-square test was used to test differences between random dataset and vectors. Bottom plots are histograms displaying the distribution of integration sites relative to gene density within a 1?Mb windows surrounding integration sites. 1742-4690-11-34-S2.pdf (653K) GUID:?CE69DE75-9180-4FB8-8050-0D8FC983D68F Additional file 3: Table S1 Vector integration sites within the tumor-associated genes (NCKU Bioinformatics Center). Integrants within the tumor-associated genes are shown. The cell color indicates whether a single or multiple integration events were detected within genes. A single integration within a gene is usually shown uncolored (name of integrant, position, strand). Two integration events of the same vector in the same gene are in yellow. Two integrations of different vectors are in orange. Red color indicates three different vectors found within the same gene. 1742-4690-11-34-S3.pdf (248K) GUID:?3CB484E2-FDA8-4F94-A8AF-F9B2391374DD Additional file 4: Table S2 Vector integrations and the tumor associated DEPC-1 genes listed in The Malignancy Gene Atlas. The same color coding as above was used. 1742-4690-11-34-S4.pdf (792K) GUID:?4204C3FF-E20C-4C3E-B470-74D59BC2961A Abstract Background Mouse.