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.