Agilent 2100 bioanalyzer high sensitivity dna kit

Isolation of RNA from brain tissue and spinal cord was performed as previously described by Stenberg et al. (2020a). The quantity and purity of the extracted RNA were measured by spectrophotometry (NanoDrop ND-1000, NanoDrop Technologies, Montchanin, DE) and Agilent High Sensitivity DNA Kit (2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA). Three of the eight healthy control piglets were excluded due to non-sufficient RNA concentration for the cDNA protocol. Synthesis of cDNA was performed using an input of 1.2 μg RNA per reaction (GoScript Reverse transcription system, Promega). To remove potential contamination of genomic DNA, the RNA was treated with RQ1 RNAse-free DNAse (Promega, Madison, WI, USA), and a -RT control was run parallel to the RNA samples. All samples were diluted 1:5 in RNase-free water and stored at − 20 °C until use.

In the MiSeq sequencing, the eight healthy piglets and six piglets with signs of splay leg were included. Sequencing libraries for the MiSeq run were prepared using the Trio RNA-Seq Library Preparation Kit (NuGEN Technologies, San Carlos, CA, USA). The library concentration was measured using a Qubit® 2.0 Fluorometer (Thermo Fisher Scientific, Paisley, UK) and an Agilent High Sensitivity DNA Kit (2100 Bioanalyzer, Agilent Technologies, Palo Alto, CA, USA). Based on the concentration, pooling and normalization to 2 nM were performed. The library pools were diluted in RNase-free water. Paired-end sequencing was performed with a MiSeq Reagent Kit v3 600 cycles on the MiSeq instrument (Illumina, San Diego, CA, USA) at the National Veterinary Institute, Uppsala, Sweden. The quality of the dataset was assessed using the FastQC software [53 ].

RNA sequencing was performed for the CDHFD (n = 3), ALS-L1023 responder (n = 3), and ALS-L1023 non-responder (n = 3) groups from the more effective dose group. A group that showed improvement in fibrosis (depending on the average of the area of fibrosis) was defined as a responder group whereas a group that did not show improvement in fibrosis was defined as a non-responder group. Libraries were prepared from total RNA using the NEBNext Ultra II Directional RNA-Seq Kit (NEW ENGLAND BioLabs, Inc., UK). mRNA was isolated using the Poly(A) RNA Selection Kit (LEXOGEN Inc., Vienna, Austria). The isolated mRNAs were used for cDNA synthesis and shearing, according to the manufacturer’s instructions. Indexing was performed using Illumina indexes 1–12. The enrichment step was performed using PCR. Libraries were subsequently checked using the Agilent 2100 Bioanalyzer (DNA High Sensitivity Kit) to determine the mean fragment size. Quantification was performed using a library quantification kit and a StepOne Real-Time PCR System (Life Technologies Inc., Carlsbad, CA, USA). High-throughput sequencing was performed as paired-end 100 sequencing using a NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA).