title: NextSeq 500 sequencing; Bulk RNAseq of anti-PD1-induced cardiac dysfunction in C57BL/6J mice
design: Bulk RNAseq of anti-PD1-induced cardiac dysfunction in C57BL/6J mice
library_name: V4_s
library_construction_protocol: Mice were sacrificed under ketamine/xylazine anaesthesia (100/10 mg/kg), underwent whole-body perfusion with phosphate-buffered saline (PBS) and hearts were stored for molecular analyses after fast freezing in liquid nitrogen. We have used the following anti-PD-1 antibody: Anti-PD-1, 200 ug / mouse, 3 times / week; BioXCell cat.no.: BP0146. For control animals, we have used the following isotype control antibody: Rat IgG2a isotype control, 200 ug / mouse, 3 times / week; BioXCell cat.no.: BP0089 Total RNA was isolated from mouse heart, thymus and spleen samples with a chloroform/isopropanol precipitation method. Briefly, Qiazol® (Qiagen, The Netherlands) was added to each sample and homogenized with TissueLyser (Qiagen, The Netherlands). Homogenates were centrifuged, and from the clean upper phase, DNA and protein were precipitated with chloroform. Total RNA was precipitated with isopropanol, and pellets were washed four times with 75% ethanol (vWR, PA, US). Finally, total RNA was resuspended in nuclease-free water. For Gene Expression Profiling (GEx) library construction, QuantSeq 3'mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen GmbH, Wien, Austria) was applied according to the manufacturer's protocol. The quality and quantity of the library was determined by using High Sensitivity DNA1000 ScreenTape system with 2200 Tapestation (Agilent Technologies, Santa Clara, CA, USA) and dsDNA HS Assay Kit with Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively.