title: RNA-seq of cancer cell collagen tracks
design: RNA-seq of cancer cell collagen tracks
library_construction_protocol: For mRNA and microRNA seq of collagen-tracks, we compared a decellularized ECM decorated with collagen-tracks to a control ECM incubated with conditioned media from the same cells. For the track-containing sample, 140,000 MDA-DDR1-GFP cells were seeded on 4 silicone dishes previously coated with 0.5mg/ml type I collagen (Corning, prepared as previously described). The control dish was coated with 0.5mg/ml type I collagen incubated with conditioned media from the same MDA-DDR1-GFP cells. After an overnight incubation, cells were detached using 12 to 16 baths of 20mM EDTA for 5 min at 37°C. The remaining cells were removed by laser microdissection using PALM type 4 microscope (Zeiss). The decellularized ECM were then lysed with 300µl Trizol and kept at -80°C before being used. RNA was extracted using the Direct-zol RNA microprep kit (Zymo Research) following manufacturer's instructions, RNA purity was assessed by capillary electrophoresis (LabChip RNA pico sensitivity assay, Perkin Elmer). For MCF-7 cells internalization mRNA-seq RNA extraction was peformed using Nucleospin RNA kit (Macherey Nagel). Small-RNA-seq libraries were synthesized using NEXTflex Small-RNA Seq kit v3 (Perkin Elmer) following the manufacturer's instructions. mRNA were reverse transcribed with an oligo-dT primer containing an adapter sequence and double-stranded cDNA was obtained with a template switching oligo (TSO) containing the same adapter sequence as the oligo-dT. After a pre-amplification with 15 cycles of PCR, we used the NEXTflex Rapid XP DNA-seq kit v2 (Perkin Elmer) to synthesize RNA-seq libraries.