title: NextSeq 500 sequencing; miRseq Analysis of Human Macrophages Co-cultured with MCF-7 Breast Cancer Cells
design: miRseq Analysis of Human Macrophages Co-cultured with MCF-7 Breast Cancer Cells
library_construction_protocol: For coculture experiments, MΦ were cultured at a density of 5 x 105 cells/mL. MCF-7 cancer cells were detached from culture flasks using trypsin-EDTA and washed with PBS. Tumor cells were resuspended in MΦ media and cocultured with MΦ at the same density. After 48 h, residual MCF-7 cells were removed from the plates using trypsin-EDTA for 3-5 min, which left adherence of MΦ unaltered.  Adhered macrophages were harvested and processed for RNA isolation according to mirVana protocol. Primary human monocytes (PBMCs) were isolated from buffy coats of anonymous healthy donors obtained from DRK Blutspendedienst using Ficoll-Hypaque gradients (PAA Laboratories). Cells were washed twice with PBS and seeded onto high-adherence culture dishes (Sarstedt, Nümbrecht, Germany). After culturing PBMCs for 1 h in RPMI 1640 supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin, non-adherent cells were washed away and remaining monocytes were cultured in media containing human serum for 7 days to allow the differentiation towards macrophages. Macrophages were either left untreated or cocultured with MCF-7 breast cancer cells for 48h. Additionally, naive macrophages were treated with 100 nM RvD1 for 6 h. We trimmed low quality regions and adapter sequences from the raw sequence reads with Trimmomatic. We first clipped leading and trailing positions with a base quality value below 3, and subsequently performed a sliding window analysis and clipped the read if a window of length 4 was encountered where the average base quality dropped below 15. Subsequently, we screened and removed adapters of the Illumina TrueSeq small RNA kit, and the DPN2 expression primer using the option ILLUMINACLIP:2:30:7 of Trimmomatic. In a second round of adapter clipping, we removed remaining short adapter sequences using the option ILLUMINACLIP:0:30:5. Sequence reads with a remaining length below 18 bp were discarded.  MΦ were harvested for RNA isolation using the mirVanaTM miRNA isolation kit (ThermoFisher Scientific, MA, USA) according to the manufacturer's instructions cDNA libraries from total RNA were generated using the TruSeq® Small RNA sample preparation protocol (Illumina, San Diego, USA). Enrichment and size distribution of the libraries were quality-assessed by capillary electrophoresis on a DNA high sensitive chip on Bioanalyzer (Agilent, Waldbronn, Germany).
attributes.tag: Experimental Factor: stimulus
attributes.value: none