GSM7806574: Mouse OTC, control-1, biol rep 1; Mus musculus; RNA-Seq
For experiments in organotypic tissue cultures, 5-6 cultures of the same mouse were collected and used per sample. For in vivo experiments, the mPFC of treated and untreated adult mice was dissected and the mPFC of the left hemisphere was used as one sample. In experiments with human material, the tissue of each patient was seen as one biological sample. RNA was isolated using the Monarch® Total RNA Miniprep Kit (#T2010S; New England Biolabs) according to the manufacturer's instructions. RNA quantity and quality were assessed using an Agilent RNA 6000 Pico Kit (#5067-1513; Agilent) with a 2100 Bioanalyzer (#G2939BA; Agilent). After RNA isolation from TTX-treated tissue cultures, library preparation and RNA sequencing was performed using the genome sequencer Illumina HiSeq technology in NovaSeq 6000 S4 PE150 XP sequencing mode (service provided by Eurofins). After RNA isolation from the mPFC of mouse in vivo experiments and human neocortical samples, poly(A)- selection was performed using a poly(A)-selection mRNA magnetic isolation module (#E7490; New England Biolabs) according to the manufacturer's instructions. For non-directional library preparation, NEBNext Ultra II RNA Library Preparation Kit for Illumina (#E7770; New England Biolabs) was used. After fragmentation and adaptor ligation, dual index primers (New England Biolabs, #E7600S) were ligated in a library amplification step using 10 PCR cycles. Libraries were finally cleaned up with 0.8X SPRI beads following a standard bead purification protocol. Library purity and size distribution were assessed with a High Sensitivity DNA assay on a Bioanalyzer instrument (Agilent). We quantified the libraries using the NEBNext Library Quant Kit for Illumina (New England Biolabs, #E7630) based on the mean insert size provided by the Bioanalyzer. A 10 nM sequencing pool (120 µl in Tris-HCl, pH 8.5) was generated for sequencing on the NovaSeq6000 sequencing platform (Illumina; service provided by CeGaT GmbH, Tübingen, Germany). We performed a paired-end sequencing with 100 bp or 150 bp read length.