Truseq adapter sequences

FastQC1 software (v. 0.11.3) was used to detect the quality control of RNA-Seq reads. With the exception of known Illumina TruSeq adapter sequences, poor reads and ribosomal RNA reads, RNA sequences were first trimmed with seqtk2 software. Then, trimmed reads were mapped to mouse reference genome by BWA-MEM software (v. 2.0.4). Next, circRNAs were predicted using CIRI software, circRNAs were matched to the circBase and terms as known circRNAs, the counts were normalized by SRPBM. The trimmed reads were also aligned to the mouse reference genome by the Hisat2 (v. 2.0.4). Stringtie (v. 1.3.0) was performed for each gene count from trimmed reads. Gene counts were normalized by trimmed mean of M-values (TMM), and fragments per kilobase of transcript per million mapped reads (FPKM) in Perl script. Differentially expressed circRNAs (DECs) between three groups were analyzed using edgeR software. Primary inclusion criteria for DECs were a FC≥2. circRNAs-miRNAs interaction was predicted by analyzing significantly dysregulated circRNAs, in accordance with Origin Biotech's custom-built software, based on miRanda software.

FastQC was conducted for Quality control (QC) of RNA-Seq reads (version. 0.11.3)¹. Trimming was performed by seqtk for known Illumina TruSeq adapter sequences, poor reads, and ribosomal RNA reads². The trimmed reads were then mapped to the Rattus norvegicus reference genome (rno6) using Hisat2 (version: 2.0.4) (Kim et al., 2015 (link); Pertea et al., 2016 (link)). Stringtie (version: 1.3.0) was performed for each gene count from trimmed reads (Pertea et al., 2015 (link), 2016 (link)). Gene counts were normalized by TMM (Robinson and Oshlack, 2010 (link)), and FPKM was performed using Perl script (Mortazavi et al., 2008 (link)). EdgeR was used to determine differential gene expression (Robinson et al., 2010 (link)), with significance determined by a p-value < 0.05 and absolute value of a log 2-fold change > 1 (Benjamini et al., 2001 (link)). Venny was applied to screen up-DEGs (MCAO vs. Control and THSWD vs. MCAO) and down-DEGs (MCAO vs. Control and THSWD vs. MCAO).

The sorted cells were centrifuged at 4000 rcf for 20 min. The supernatant was decanted carefully and the tubes were inverted over a paper towel to remove excess liquid (note that the pellets were not visible). The cell pellet was resuspended with 50 μL of water by vigorous pipetting in the bottom of the conical tube. The suspension was transferred to a microcentrifuge tube and boiled for 10 min to lyse the cells. The lysate was centrifuged at 13,000 rcf, then 10 μL of the supernatant was used as the template in a 50 μL PCR reaction to amplify the peptide genes with flanking Illumina TruSeq adapter sequences. After amplification, the PCR reaction mixtures were diluted 100-fold, and this diluted solution was used as a template for a second round of PCR to append unique 5’ and 3’ indices for each sample. Multiple samples from different experiments were pooled and sequenced on an Illumina MiSeq system using standard protocols for paired-end sequencing. Typically, DNA was loaded to obtain at least 500 read counts for each variant in an unsorted library and equivalent total read counts for all of the variants in unsorted and sorted libraries.