Superscript 3

RNA was extracted from the OS cells by RNAeasy^TM Plus Animal RNA Isolation Kit with Spin Column (Beyotime, Jiangsu, China). cDNA was synthesized with SuperScript III (Takara, Dalian, China) according to the manufacturer's protocol. Real-time PCR analysis was performed using the Applied Biosystems 7500 Real-Time PCR System, according to the manufacturer's instructions. The reactions were performed for three independent experiments and results were normalized to β-actin. The primer sequences used can be found in the Table S2. The mean±SD of three independent experiments is shown.

Total RNA was extracted from CFs by TRIzol (Invitrogen, USA). cDNA was synthesized using SuperScriptIII (Takara, Japan). cDNA was amplified by use of SYBR Green (Takara, Japan). Relative gene expression levels were quantitatively assessed using the 2^-△△CT method. Primers for PRR and the housekeeping gene GAPDH were as follows: rat PRR forward 5′-TCTGTTCTCAACTCGCTCCC-3 and reverse 5′-TCTCCATAACGCTTCCCAAG-3′ and rat GAPDH forward 5′-TCTCTGCTCCTC CCT GTTCT-3′ and reverse 5′-ATCCGTTCACACCGACCTTC-3′.

Protein–protein interaction data for prostate cancer-related genes were obtained from the STRING database. A network was visualized using the software Cytoscape (3.9.1 version; https://cytoscape.org/index.html)^{123 (link)}, and the core interacting protein network was extracted.
Total RNA from all samples was extracted using the TRIzol reagent (Invitrogen, USA), and genomic DNA contamination was removed using DNase I (Promega, USA). RNA yield was measured using a NanoDrop ND-2000 (Thermo Fisher Scientific, USA), and integrity was assessed on a 1% agarose gel. cDNA was synthesized using oligo-dT primers and SuperScript III (Takara, JP). Real-time Quantitative PCR (RT-qPCR) was performed using the Plus real-time PCR system (Applied Biosystems, USA). mRNA RT-qPCR was carried out using the SYBR Prime ScriptTM RT-PCR kit (Takara, JP). Data were analyzed using the relative 2^−ΔΔCT method^{124 (link)}. RT-qPCR primer sequences are listed in Supplementary Table S22.

Smart-seq2 protocol for single nuclei sequencing was performed as described in^{60 (link)} with several modifications^{61 (link)}. In brief, individual nuclei were lysed, RNA lysate was reverse transcribed with 100U of Superscript III (Takara) with the addition of 10U of RNaseIn, 5 mM DTT, 1 M betaine (Sigma), and 6 mM MgCl2 (Invitrogen). Reverse transcription reaction was paused after 90 min at 42 °C for adding TSO oligos at the final concentration of 0.5 uM and continued for additional 12 min. The cDNA was amplified using KAPA HotStart HIFI 2 × ReadyMix (Kapa Biosystems) and 10 uM ISPCR primer for 24 cycles (98 °C – 3 min; 10 cycles: 98 °C – 20 s, 60 °C – 1 min, 72 °C – 6 min; 7 cycles: 98 °C – 20 s, 64 °C – 1 min, 72 °C – 6 min; 7 cycles: 98 °C – 20 s, 67 °C – 1 min, 72 °C – 6 min; 72 °C – 10 min; 4 °C – hold). Amplicons were purified by means of Ampure XP beads (Beckman Coulter, USA) at a sample:bead ratio of 1:1, their concentration was assessed using Qubit dsDNA HS Assay Kit (Invitrogen), while the amplicon quality was estimated by cDNA length on high-sensitivity DNA chip (Agilent) on Agilent 2100 Bioanalyzer. Oligo-dT, TSO and IS PCR primers (sequences are listed in Table S2) were ordered from Exicon (http://www.exiqon.com/). All primer sequences are listed in the Table S1.

The TRIzol solution (Qiagen, Switzerland) was used to extract the total RNA from the HUVECs according to the instructions of the manufacturer, followed by transcribing the RNAs to cDNA using the SuperScript III (Takara, Tokyo, Japan) reagent. The SYBR green quantitative PCR kit (Takara, Tokyo, Japan) was used to determine the relative expression of target genes, while GAPDH was taken as the negative control. QRT-PCR was performed on the ABI Prism 7500 Fast Sequence Detection System (Applied Biosystems, CA, USA). The 2^-ΔΔCt method was used to calculate and quantify the relative expression level of target genes. The following primers were used in this study: LOX-1 (F: 5’-TGATAGAAACCCTTGCTCG-3’, R: 5’- TTGCTTGCTGGATGAAGTC-3’); MCP-1 (F: 5’-GTTGGCTCAGCCAGATGCA-3’, R: 5’-AGCCTACTCATTGGGATCATCTTG-3’); CXCL-2 F: 5’-GGCAGAAAGCTTGTCTCAACCC-3’, R: 5’-CTCCTTCAGGAACAGCCACCAA-3’); eNOS (F: 5’-GTGGCTGTCTGCATGGACCT-3’, R: 5’-CCACGATGGTGACTTTGGCT-3’); Occludin (F: 5’-TACAGCAATGGAAAACCACACT-3’, R: 5’-CAAAGGAATGGGAAACGACTAA-3’); KLF2 (F: 5’-CCAAGAGTTCGCATCTGAAGGC-3’, R: 5’-CCGTGTGCTTTCGGTAGTGGC-3’); GAPDH (F: 5’-GACGGCCGCATCTTCTTGT-3’, R: 5’-CAGTGCCAGCCTCGTCCCGTAGA-3’).

For cells and tissues, total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, US) in accordance with the manufacturer’s instructions. One microgram of total RNA was reverse transcribed to generate cDNA with Superscript III (Takara, Kusatsu, Shiga, Japan). RT-qPCR was performed using Premix Ex Taq (Takara, Kusatsu, Shiga, Japan) by a Step One Real-Time PCR System (Bio-Rad). The target genes were normalized to the housekeeping gene (Hprt or Gapdh). Fold changes were presented as a result of 2^-ΔΔCt. Relative gene expressions were presented as a result of 2^-ΔCt. Primer sequences were based on Primer-Bank (http://pga.mgh.harvard.edu/primerbank) and blasted to confirm the target genes using Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast). The primer sequences used are shown in Supplementary information, Table S1.