RNA extraction, RNA-sequencing library, and comparative
real‑time PCR analysis
Total RNA was isolated from BMMNCs using the TriPure reagent (QIAGEN,
Netherlands) according to the manufacturer’s instructions. RNA-seq
libraries were manufactured by the KAPA HyperPrep kit with RiboErase and
the RNA-seq was performed by 100 million paired-end reads on an Illumina
NovaSeq 6000 platform (CeGaT company, Tubingen, Germany). Additionally,
total RNA was treated with DNase I (Thermo Fisher Scientific,
Waltham, MA) to eliminate genomic DNA, and the first strand cDNA was
synthesized using the First Strand cDNA Synthesis Kit (AddScript cDNA
Synthesis kit) according to the manufacturer’s protocol. The mRNA
expression level of SPRING1 (or C12orf49) was determined by
comparative real-time PCR using SYBR Green Master Mix (AMPLIQON,
Denmark) on a CFX96 BioRad System thermocycler (Roche, Germany) with
specific primers (forward primer 5′-CCCAACAAGCAACTTCTC-3′ and reverse
primer 5′-TCTCCATAGCAATACTTTGC-3′) through calculating theSPRING1 mRNA expression level using the 2-ΔΔCtmethod and beta-glucuronidase (GUSB ) as an endogenous control.