Collibri stranded rna library prep kit

Total RNA of kidneys from three rats was extracted with TRIzol (Invitrogen, #15596026). The mRNA was purified from total RNA by an mRNA Purification Kit (Invitrogen, #61006) and was converted to double-stranded cDNA. The cDNA library was generated using the Collibri Stranded RNA Library Prep Kit (Invitrogen, #a38994024). All samples were sent to the GENEWIZ Technology Corporation (SuZhou), and an Illumina platform was used for mRNA sequencing.
The quality of raw sequencing data was assessed with FastQC, which provided a quality profile such as GC content biases, overrepresented sequences, and adapter content. The low-quality reads were then trimmed and filtered by Trimmomatic^{35 (link)}. The clean reads were mapped to the Rattus norvegicus reference genome (Rnor_6.0) using STAR spliced read aligner^{36 (link)}, and the Rsubread package in R, which contains the feature Counts function^{37 (link)}, was applied to determine count matrices of gene counts. The fold change in the gene expression level between the sham and UUO samples was estimated using the GFOLD algorithm^{38 (link)}.

Laccaria bicolor RNA was extracted using RNAzol RT Reagent (MRC), treated with DNaseI (Thermo Scientific) as recommended by the vendor and the longer than 200 nt fraction was purified using RCC (Zymo Research). Then, ribosomal RNAs were depleted using a Ribo-Zero Gold rRNA Removal Kit (yeast; Illumina) according to recommendations of the vendor and the longer than 200 nt fraction was purified using RCC (Zymo Research). RNA libraries were further prepared and sequenced at Thermo Fisher Scientific Baltics, using a Collibri Stranded RNA Library Prep Kit (Invitrogen) for Illumina (approx. 200 M pair-end reads per sample).

Total RNA was isolated from PBMCs using Isospin cell and tissue RNA kit (Nippon Gene) or an RNAdvance v2 kit (Beckman Coulter) according to manufacturer instructions and quantified with an RNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher). For transcriptome analysis, 10 ng of RNA were used for library preparation with a QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen) according to the manufacturer’s protocol for low-input RNA samples. To generate single-nucleotide polymorphism (SNP) calls for several donors whose samples were analyzed by scRNA-seq, cDNA libraries were prepared from 500 ng of RNA using a Collibri Stranded RNA Library prep Kit (Thermo Fisher) according to the manufacturer’s protocol for degraded RNA samples. Libraries were quantified with a Qubit 1x dsDNA HS Assay Kit (Thermo Fisher) and a Qubit Flex Fluorometer (Thermo Fisher), and quality was assessed using D1000 ScreenTape and High Sensitivity D5000 ScreenTape with a Tapestation 2200 (Agilent). Pooled libraries were sequenced on a Novaseq 6000 instrument (Illumina) with 1 × 100-bp reads for transcriptome analysis and 2 × 150-bp reads for generation of SNP calls at the Sequencing Section at OIST.