High-throughput RNA-seq
RNA library preparation was conducted by a kit of
Truseq® chain total RNA sample preparation (Illumina,
USA) based on provided directions, with the final library being
evaluated with a Qubit 2.0 fluorometer and an Agilent 2100 Bioanalyzer
for quantification and quality control. CBOT was then used to dilute the
library to 10 pm for cluster generation, after which an Illumina Hiseq
2500 instrument (Illumina) was used for sequencing. All library
construction and sequencing were performed by OG-Biotech Inc. (Shanghai,
China).
For data analysis, RNA-seq read data were first subjected to quality
control (QC) using FastQC (v. 0.11.3)
(http://www.bioinformatics.babraham.ac.uk/projects/fastqc ), after
which low-quality reads, rRNA sequences, and adapter sequences were
trimmed with the seqtk tool (https://github.com/lh3/seqtk ).
Mapping to the hg38 human reference genome was then performed with the
BWA-MEM computer program (v 2.0.4), circRNAs were estimated by utilizing
CIRI (33), while SRPBM was used to determine circRNA counts(34).
Screening for circRNAs that were differentially expressed in AS and
control samples was conducted utilizing edgeR using the following
criteria: FC ≥ 2 or < 0.5 and P -value < 0.05.
Functional characterization of circRNAs of interest was performed
through GO and KEGG enrichment analyses of the parental genes from which
these circRNAs were derived. The miRNA targets of these DECs were
predicted with the miRanda software-based custom-built software
application used by OG-Biotech(35), and Cytoscape was used for
circRNA-miRNA network visualization(36).