GSM4983271: UT-SCC-8 shANO1_65_1; Homo sapiens; RNA-Seq
Cell suspension of 500 µl (concentration of 300 000 cells/ml) were suspended in collagen I matrix (500µl) and plated in low adhesion plates (poly-HEMA coated to prevent cell adheresion on the 24-well plates, Corning), and grown as inert in collagen I for five days. To extract the RNA, media was removed from the well, cultures were washed three times with PBS and Trizol (Ambion, Life Technologies) was added on top of the collagen and homogenised by precellys ceramic beads and homogenizator (Precellys). Briefly, RNA was isolated using chloroform/ isopropanol based method and purified with Qiagen RNeasy Mini kit, according to manufacturer's instructions (Qiagen). 1µg of total RNA was subjected to library synthesis. Libraries were prepared according to Illumina's instructions accompanied with RiboZero Complete Gold Human and NEBNext Directional RNA library kit. Library quality was checked by Bioanalyzer analysis (Agilent 2100). Library denaturation and dilution was made according to NextSeq500 system guide.