Qiaseq fastselect rrna hmr

RNA was isolated by use of an RNeasy FFPE Kit (Qiagen), and deparaffinization was performed using 640–750 µL deparaffinization solution from the kit with 60–90 min incubation at 56 °C. RNA was eluted with 2′ 14 µL of RNase-free water. rRNA was removed by use of a QIASeq FastSelect (rRNA HMR, Qiagen), using 1 µg RNA or as much as could be handled by the kit (max 10 µL). RNA-sequencing libraries were constructed with a TruSeq Stranded mRNA Library Prep kit (Illumina) and 0.3 µL adapters with 30 PCR cycles. Barcodes were optimized by use of BARCOSEL software (http://ekhidna2.biocenter.helsinki.fi/barcosel/)^{34 (link)}. Sequencing was performed using an Illumina Novaseq 6000 instrument. Adapter sequences, low-quality bases (q = 25), and short sequences (m = 30) were first trimmed using cutadapt (v.4.1)^{35 (link)} (https://cutadapt.readthedocs.io/en/stable/). The fourteenth (p14) patch release for the GRCh38 reference assembly and annotation was downloaded from https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/001/405/GCF_000001405.40_GRCh38.p14/. The STAR aligner (v2.7.9a_2021-06-25) (https://code.google.com/archive/p/rna-star/) with default parameters was used to map reads against the reference sequence^{36 (link)}. Sorting and indexing of the alignment files were performed using Samtools (v.1.10) (https://www.htslib.org/)^{37 (link)}.

The concentration of the total RNAs was measured by using DeNovix fluorometer (DeNovix). Sample purity was checked by using Nanodrop (Thermofisher). Integrity of the total RNAs was assessed by using Agilent 2100 Bioanalyzer (Agilent). Approximately 1 μg of the total RNAs from each sample was used to create individually indexed strand-specific RNA-Seq libraries by using QIAseq FastSelect RNA removal and QIAseq stranded total RNA library preparation kits (QIAGEN). Eukaryotic and prokaryotic rRNAs were removed by adding 1 μL of QIAseq FastSelect –rRNA HMR (Human, mouse, rat) and QIAseq FastSelect –5S/16S/23S-rRNA (bacteria) removal solution (QIAGEN, USA), and the reactions were subjected to fragmentation and cDNA synthesis. AMPure XP beads (Beckman Coulter Genomic) were used to separate the cDNAs from the reaction mix. Indexing adapters were ligated to the cDNAs, and subsequently all cDNA libraries were inspected for quality by using Agilent 2100 Bioanalyzer (Agilent) and quantified with DeNovix fluorometer (DeNovix). The indexed sequencing libraries were pooled in equimolar quantity and subjected to cluster generation and paired-end 2 × 150 nucleotide read sequencing on Illumina Hiseq sequencer.

Total RNA was extracted from whole blood with the QIAmp viral RNA extraction kit and eluted in pure water. Following the manufacture’s protocols, total RNA was used as input material into the QIAseq FastSelect–rRNA HMR (Qiagen) protocol to remove cytoplasmic and mitochondrial rRNA with a fragmentation time of 7 or 15 min. Subsequently, the NEBNext^® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs) was used to generate the RNA libraries, followed by 11 or 13 cycles of amplification and purification using AMPure XP beads. Each library was quantified using Qubit and the size distribution assessed using the Agilent 2100 Bioanalyser and the final libraries were pooled in equimolar ratios. The raw fastq files (2 ×150 bp) generated by an Illumina® NovaSeq 6000 (Illumina®, San Diego, USA) were trimmed to remove Illumina adapter sequences using Cutadapt v1.2.1⁵⁴ . The option “−O 3” was set, so the that 3’ end of any reads which matched the adapter sequence with greater than 3 bp was trimmed off. The reads were further trimmed to remove low quality bases, using Sickle v1.200⁵⁵ with a minimum window quality score of 20. After trimming, reads shorter than 10 bp were removed. Sequence reads that mapped to Macaca mulatta or Macaca fascicularis are deposited under NCBI bioproject: PRJNA681111.