Universal rna extraction kit

Total RNA was extracted from human spermatozoa using a universal RNA extraction kit (TIANGEN Biochemical Technology Co. Ltd., Beijing, China) according to the manufacture’s protocol. Subsequently, cDNA was obtained by reverse transcription using a commercial kit (TIANGEN Biochemical Technology Co. LTD., Beijing, China). Abundance of mRNA transcripts encoding iPLA 2β, P53, Zinc finger E-box binding homeobox 1 (ZEB1), GPX4, and GAPDH were measured by quantitative real-time PCR. Primer sequences are shown in Table 1.

All gene manipulation was based on the genome of L. plantarum K25 in NCBI. RNA was extracted using a Universal RNA Extraction Kit (TianGen, China) according to the instructions. RNA yield and quality were evaluated with a UV spectrometer Q5000 (Quwell). Then, approximately 4 ul of RNA was reverse transcribed with a Tastline Cell DNA Kit (TianGen). Quantitative real-time PCR (qRT-PCR) was performed using the Real Master Mix (TianGen) with a final volume of 25 ul in each reaction system, which included 12.5 ul Mix containing SYBR Green 1, dNTPs, AmpliTaq Gold Fast DNA polymerase LD, 10x buffer, and 1 ul cDNA, 0.5 ul of each primer and 10.5 ul of ddH₂0, the target genes involved in the genome sequence of L. plantarum K25 and primers (designed by Primer Primer 5.0). The amplification procedure included: denaturation at 94°C for 2 min; 35 cycles of 94°C for 30 sec; 53°C for 30 sec; 72°C for 30 sec, and 72°C for 10 min. At the end of PCR cycles, melting curve analyses were performed using the LightCycle Nano qRT-PCR system. All the samples were produced and run in triplicate, and 16S rRNA gene was used as the reference. The primer sequences for the representative proteins highly differentially expressed were listed in Table S1.

The total RNA of each sample was extracted using a universal RNA extraction kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer’s instructions. RNA concentration and quality were determined with a Qubit 2.0 fluorometer (Life Technologies, Carlsbad, CA, USA), and a spectrophotometer (NanoPhotometer; Implen, Calabasas, CA, USA), respectively. RNA integrity was measured using a Bioanalyzer 2100 system with the RNA 6000 Nano Assay kit (Agilent, Carlsbad, CA, USA).
Nine strand-specific RNA libraries were prepared with an insert size of ~250–500 nucleotides using a UTP method (Parkhomchuk et al., 2009 (link)), and then were sequenced by Biomarker Technologies Corporation (BMK, Beijing, China) on the Illumina HiSeq 2000 platform with the 150-bp paired-end method and a sequencing depth of ~53 million reads per library (Table 1).