GSM5014985: Microdissected paraffin-embedded (FFPE) tongues of CBA mice, Normal  stroma, biological replica 3; Mus musculus; RNA-Seq
To ensure RNase-free conditions, only RNase-free solutions and materials were allowed. RNAse-free MembraneSlide1.0 PEN (Zeiss®) for preparation of FFPE-samples were used. To overcome the hydrophobic nature of the membrane, the MembraneSlide were initially irradiated with UV light at 254 nm for 30 min (in a cell culture hood), in order to improve FFPE-sections adhesions. Positive side effects were sterilization and destruction of potentially contaminating nucleic acids5. Unstained 3-µm sections were then mounted onto MembraneSlides the same way as routinely done using glass slides. Four to six sections were mounted on the same membrane slide for reasons of simplicity, efficiency and expediency (Supplementary Figure 2). The slides were let dry overnight in a drying oven at 56°C. To allow laser cutting and lifting, a coverslip and standard mounting medium was not applied.  Laser capture microdissection (LCM) was performed the following day. Because paraffin reduces laser efficiency (sometimes completely inhibiting cutting and lifting), we removed the paraffin before staining, laser cutting and lifting. Our deparaffinization protocol of unstained sections was the following: xylene 1.30 minute, 100% then 96% to finish 70% ethanol each 1 minute. After deparaffinization, the mounting sections were stained with RNAse free cresyl violet to preserve RNA quality (cresyl violet for 15 seconds, removing excess stain, 70% ethanol 30 sec and 100% ethanol 30 sec). After drying for 2 minutes, membrane slides were immediately used to avoid RNA degradation of deparaffinized samples. The selection of epithelial cells as well as the underlying stroma and muscle of interest was performed according to the mapping provided by the pathologist (A.T). Microdissection should be performed at least at a magnification of about 10× to respect histologic details. The energy cut should be among 50 to 60% to allow efficient cutting without risk of burning of the targeted tissue. Microdissection of the stroma should always be realized before the epithelium to avoid stromal damages (Supplementary Video). One hour of LCM allows the capture of 1mm2 and 0.3 mm2 of epithelium and stroma respectively. Microdissected specimens were collected in Adhesive Cap500® tube. We performed total RNA extraction using QIAGEN RNeasy® FFPE-kit. At the end of the extraction protocol, aliquots of 2µL were used to control quantity as well as to assess the RNA purity (Thermo-Scientific™ NanoDrop 2000). Other aliquots of 2μL were submitted to the High Sensitivity RNA Screen system (Agilent 2200 Tapestation®) allowing measurement of the RNA integrity number (RIN) and the percentage of RNA fragments >200 nucleotides (DV200) which is a more reliable determinant of RNA quality for library preparation (TruSeq stranded total RNA Library Preparation Kit with Ribo-Zero Illumina®). RNA samples with DV200 < 30% were not sequenced. After the quality control steps (qPCR, Tapestation®), sequencing was performed using the NextSeq 550 system (Illumina®). After total RNA extraction (QIAGEN RNeasy® FFPE-kit), samples (n=13 epithelial and n=13 stromal, subsequently referred as E-samples and S-samples respectively) were sequenced (NextSeq 550 Illumina®) (quality controls in Supplementary Table 1).