title: Illumina NovaSeq 6000 paired end sequencing; RNA-Sequencing of USP15 knock-out mice injected with kainate
design: RNA-Sequencing of USP15 knock-out mice injected with kainate
library_construction_protocol: Unilateral, intrahippocampal kainic acid (ihKA) or NaCl injections were performed in adult male USP15-/- and WT littermates. Mice were anesthetized and received analgesic treatment (100 mg/kg ketamine, 5 mg/kg xylazine, 0.1 mg/kg atropine, i.p. and 4 mg/kg carprofen, s.c) and placed into a stereotaxic frame (David Kopf Instruments, CA, USA) in flat skull position. KA solution (50nl, 20 mM in 0,9% sterile NaCl, Tocris) was injected into the right dorsal hippocampus (coordinates from bregma (in mm): anterioposterior (AP) = -2.0, mediolateral (ML) = -1.4, dorsoventral (DV) = -1.9) with a metal cannula (Harry Rieck Edelstahl) connected to a Hamilton syringe (Hamilton Bonaduz AG) and a micropump. Controls received NaCl solution only. After injection, mice were transferred to their home cage and kept under video surveillance for at least 8h (mean 18.1h, range 8-23h). Mice were then kept in groups in their home cages and perfused at 4d after KA injection. Mice were anesthetized with ketamine (100 mg/kg) and medetomidine (Dormitor; 1 mg/kg) and transcardial perfusion was performed with 30 ml phosphate buffered saline (PBS, ph 7.4). Brains were excised immediately afterwards and both hippocampi were prepared in ice-cold PBS, snap frozen in individual RNAse-free, sterile tubes in liquid nitrogen and stored at -80°C until all samples were collected. Total RNA was extracted from both left and right mouse hippocampi using RNeasy Plus Mini Kit (Qiagen, 74134) following manufacturer's instructions. RNA concentration was measured with a NanoDrop ND-1000 Spectrophotometer. RNA quality was assessed using the Bio-Rad Experion Automated Electrophoresis System (RQI ≥ 7.3). The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina was used to process the sample(s). The sample preparation was performed according to the protocol \"NEBNext Ultra II Directional RNA Library Prep Kit for Illumina\" (NEB #E7760S/L). Briefly, mRNA was isolated from total RNA using the oligo-dT magnetic beads. After fragmentation of the mRNA, a cDNA synthesis was performed. This was used for ligation with the sequencing adapters and PCR amplicification of the resulting product. The quality and yield after sample preparation was measured with the Fragment Analyzer (see appendices). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp).