Nanodrop 2000

Total RNA of cells was harvested with TRIzol (Takara, Terra Bella Ave, CA, USA) and quantified by a NanoDrop 2000. RNA integrity and gDNA contamination were tested by denaturing agarose gel electrophoresis (Thermo Fisher Scientific, Waltham, MA, USA), and the sequencing library was determined by an Agilent 2100 Bioanalyzer using an Agilent DNA 1000 chip kit (Agilent, Santa Clara, CA, USA). RNA-seq analyses were performed on HiSeq 4000 platforms (Illumina, San Diego, CA, USA). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis is a functional analysis mapping genes to KEGG pathways and was performed by DAVID^{29 (link)}. Heatmap was built using pheatmap in the R library.

Total RNA was extracted from all the samples using Trizol reagent. After measuring the RNA concentration using Nanodrop 2000, 2 μg RNA was reverse transcribed into cDNA using a kit from Takara Co. (RR003A; Japan). The cDNA obtained by reverse transcription was subjected to real-time fluorescence quantification of gene expression on StepOnePlus™ Real-Time PCR System (Applied Biosystems; Shanghai, China) using a Realtime PCR Master Mix kit (QPK-201; Toyobo, Japan). The PCR thermal cycles comprised initial denaturation at 95°C for 5 min; followed by 40 cycles of 15 sec at 95°C, 1 min at 60°C and 30 sec at 72°C; and a final extension for 10 min at 72°C. At the end of the PCR reaction. CT values were derived from the PCR software for relative quantification using the 2^−ΔΔCT method [29 (link)]. GAPDH/U6 was used as an internal reference. Primer sequences were listed as following:
TTTY15, forward 5′-TGAGGGAGGGAT GTAGCTTT-3′;
reverse 5′-GAAGTCAAGCAGGCAACTGA-3′;
miR-98-5p, forward 5′-TGAGGTAGTAGTTTGTGCTGTT-3′;
reverse 5′-GCGAGCACAGAATTAATACGAC-3′;
GAPDH, forward 5′-CATCATCCCTGCCTCTACTGG-3′;
reverse 5′-GTGGGTGTCGCTGTTGAAGTC-3′;
U6 S 5′-GGAACGATACAGAGAAGATTAGC-3′;
Stem-loop-R 5′-CTCAACTGGTGTCGTGGAGTC-3′;
PCNA, forward 5′- CCTGCTGGGATATTAGCTCCA-3′;
reverse 5′-CAGCGGTAGGTGTCGAAGC-3′;
CCND2, forward 5′-ACCTTCCGCAGTGCTCCTA-3′;
reverse 5′-CCCAGCCAAGAAACGGTCC-3′.

Total RNAs from cells and tissues were extracted with TRIzol reagent (Thermo Scientific, NY, USA). RNAs were quantified by NanoDrop 2000 and a total of 1mg RNA were reversely transcribed with Prime Script TM Master Mix (TakaRa, Dalian, China). qRT-PCR was performed with SYBR Premix EX Taq TM II (Takara) on ABI7500 (Thermo Fisher Scientific, CA, USA). GAPDH was included as an inner control. The qPCR protocol was shown as below: initial denaturation at 95°C for 5 min, followed by 45 repeats of a three-step cycling program consisting of 10 sec at 95°C (denaturation), 10s at 60°C (primer annealing), and 10 sec at 72°C (elongation), and a final extension step for 10 min at 72°C. The primers used were listed here: WIF1: Forward: 5'-TCTGGAGCATCCTACCTTGC-3' and Reverse: 5'-ATGAGCACTCTAGCCTGATGG-3'; SFRP: Forward: 5'- CGTGGGCTCTTCCTCTTCG-3' and Reverse: 5'- ATGTTCTGGTACTCGATGCCG-3'; DKK1: Forward: 5'- CTCATCAATTCCAACGCGATCA and Reverse: 5'- GCCCTCATAGAGAACTCCCG-3'; GAPDH: Forward: 5'- GTGGACATCCGCAAAGAC-3' and Reverse: 5'- AAAGGGTGTAACGCAACTA-3'.