Unknown,Transcriptomics,Genomics,Proteomics

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RNAseq of iTAM and mTAMs isolated from tumors in Junf/f and Jun Csf1r KO mice

ABSTRACT: Tumor-associated macrophages (TAMs) play important roles in cancer progression and resistance to therapy. Recent studies have shown that TAMs include both long-lived resident tissue macrophages (RTMs) and short-lived monocyte-derived macrophages (MDMs) with limited proliferative potential. RTMs and MDMs have been suggested to play divergent roles in tumorigenesis; RTMs are aligned with trophic functions, whereas MDMs are enriched for immune-regulatory pathways. Here we established a specific role for the AP-1 factor JUN in the differentiation and maintenance of MDMs and the specification of pro-tumoral trophic functions during tumor development. Alternatively, the immune-regulatory functions of TAMs remained JUN-independent. JUN was required for the specification and maintenance of pro-tumoral TAMs that support blood vessel maturation and tumor growth. Single-cell transcriptomics analysis uncovered the alternative fates for tumor-infiltrating monocytes and the development of distinct TAM states associated with trophic functions and immune-regulation. These studies demonstrate an important role for JUN in the specification of pro-tumoral monocyte-derived TAMs that could offer opportunities for selective TAM-targeted therapies for cancer.

INSTRUMENT(S): Miltenyi Biotec (cat no. 130-096-730) ,Fortessa X-20 flow cytometer (BD Biosciences), R software, nf-core pipeline, Illumina NovaSeq 6000, Illumina DNA Tagmentation, Arcturus PicoPure

ORGANISM(S): Mus musculus

SUBMITTER: Abhinav Soni

PROVIDER: E-MTAB-17032 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Arginine-rich mixed charge domains (R-MCDs) contribute to and alter the properties of nuclear speckles. We are interested in how this affects the retention of poly-adenylated mRNAs in the nucleus. This experiment tests how the expression of the R-MCD of PPIG influences the nuclear-cytoplasmic distribution of mRNAs over time. Specifically, a HeLa cell line in which PPIG expression is driven by a doxycycline-inducible promoter was used, and doxycycline was added for 0, 4, 8 or 12 hours. This time course was performed in triplicates. Nuclear and cytoplasmic fractions were collected from the cells and RNA was extracted. 3' end sequencing libraries were produced by fragmenting the RNA and introducing Illumina adapters via a oligo-dT-primed reverse transcription and template switching oligo approach. The data indicate that mRNAs containing long, multivalent GA-rich regions in their coding sequences are more retained in the nucleus over time following expression of the R-MCD. The files provided in this accession have not been trimmed to remove adapters, 3' poly-A sequences, UMIs or G-stretches from TSOs. The data was analysed using nf-core/rna-seq using the following command: nextflow run nf-core/rnaseq \\ --input samplesheet.csv \\ --fasta '/camp/lab/ulej/home/users/farawar/genomes/hs/fasta/GRCh38.primary_assembly.genome.fa' \\ --gtf '/camp/lab/ulej/home/users/farawar/genomes/hs/annotation/gencode.v29.annotation.gtf' \\ --salmon_index '/camp/lab/ulej/home/users/farawar/genomes/hs/salmon_index/salmon_index' \\ --gencode \\ --pseudo_aligner salmon \\ --with_umi \\ --umitools_bc_pattern NNNNN \\ --clip_r1 5 \\ -resume \\ -profile crick \\ --outdir nf-core-results \\ -c extraconfig.config With the extra config file containing: withName: '.*:QUANTIFY_STAR_SALMON:SALMON_QUANT' { ext.args = '--noLengthCorrection' } Therefore removing the first 5 nucleotides as UMI sequences and the following 5 nucleotides as they contain G-stretches from the template-switching oligo.

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RNAseq of iTAM and mTAMs isolated from tumors in Junf/f and Jun Csf1r KO mice

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