title: Transcriptome analysis of cuprizone mouse model following AAV-mediated delivery of A Proliferation Inducing Ligand
design: Transcriptome analysis of cuprizone mouse model following AAV-mediated delivery of A Proliferation Inducing Ligand
library_construction_protocol: For each of the 6 experimental groups, transcriptomic analysis was performed separately for the dissected splenium and cortex. Each group contained 5 biological replicates that were sequenced once (total of 60 sequencing datasets).  Total RNA was isolated using the RNeasy® Mini kit (Qiagen, Benelux, Belgium), according to the manufacturer's protocol. Briefly, tissue was disrupted and lysate homogenized with lysis buffer containing b-mercaptoethanol, followed by RNA extraction and purification using RNeasy Mini spin columns. The concentration of the RNA samples was measured on the Qubit fluorometer 4.0, using the Qubit high sense RNA assay (Thermo Scientific, Wilmington, USA). The integrity and purity of the RNA samples was evaluated by the Agilent 4150 TapeStation System using the recommended RNA ScreenTape assay (Agilent Technologies, Inc., Waldbronn, Germany). For all samples, the RNA integrity number (RIN) values were above 7, indicating the superior quality of the isolated RNA.  The mRNA library preparation was performed using the QuantSeq 3' mRNA-Seq V2 Library Prep Kit FWD with UDI 12 nt Set B1, combined with the QuantSeq-Flex First Strand Synthesis Module V2 to create longer fragments (Lexogen, Vienna, Austria). Before the library amplification PCR, the optimal PCR cycli were evaluated with the PCR Add-on and Reamplification Kit V2 (Lexogen, Vienna, Austria). The finalized cDNA libraries concentrations were measured using the Qubit high sens dsDNA kit (Qubit 4.0, Thermo Scientific, Wilmington, USA). The fragment lengths, distribution and the quality of the libraries were evaluated using the D1000 ScreenTape assay (TapeStation, Agilent Technologies, Inc., Waldbronn, Germany).