GSM5767619: Non Tumor region 11; Homo sapiens; RNA-Seq
Serial 10 µM sections of human liver tissue were mounted on PET-membrane steel frame slides (Leica) and stored at −80 °C until use. For laser dissection, slides were fixed for 20 min in 70% ethanol at +4 °C followed by dissection using a PALM MicroBeam laser UV dissector (Zeiss). Serial section alignment of the smiFISH and unstained slides was carried out at the Laser Microdissection platform at Bordeaux Neurocampus using an automated serial section alignment and microdissection (PALM 4 technology, Zeiss). RNA was extracted and purified from each sample (circa 100–200 cells) using the RNeasy® Micro kit (Qiagen) with on-column DNAse I digestion. RIN values were calculated using the Agilent Bioanalyzer with RNA pico chips. RNA samples containing a minimum of 1 ng RNA and a RIN >4 were used for further RNAseq analysis. RNA was amplified using the Ovation® SoLo RNA-seq library preparation kit (NuGEN) used for low input or single-cell sequencing according to manufacturer's instructions, including a ribosomal RNA depletion step (Insert Dependent Adaptor Cleavage). Libraries were then monitored for concentration and fragment sizes using a Fragment Analyzer (kit Standard Sensitivity NGS) and by qPCR (ROCHE Light Cycler 480) prior sequencing on Illumina HiSeq2000 platform (single end 50 bp length).