Eastep rt master mix

Total RNA was extracted from 7 PB specimens with FastPure^® Cell/Tissue Total RNA Isolation Kit (Vazyme Biotech Co., Ltd., Nanjing, China) and then reverse-transcribed to cDNA with HiScript II Q RT SuperMix (+gDNA wiper) (Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s instructions. The generated cDNAs were amplified in the subsequent qRT-PCR using Eastep^® RT Master Mix (Promega, Beijing, China). The gene expression levels were calculated by the 2ΔΔCT method and human GAPDH was used as a housekeeping gene to normalize target genes. Primers used in real-time PCR are available in the Supplementary Table S2.

Genomic DNA was purified using a commercial DNA extraction kit (TIANGEN, Beijing, China). Samples were then treated with sodium bisulphate to convert unmethylated cytosine to uracil using an EZ DNA Methylation Kit (Zymo Research, Irvine, CA, US). The bisulphite transformed DNA was used to amplify the product in the MMP2 promoter area, and the level of MMP2 methylation level was represented by the percentage of methylated CpG in methylated unmethylated CpG using qRT-PCR assays.
For qRT-PCR, RNA was isolated with RNAprep pure Cell/Bacteria Kit (TIANGEN) and reverse-transcribed with the GoScript™ Reverse Transcription System (Promega). The collected cDNA and genomic DNA collected in other experiments were amplified using Eastep® RT Master Mix (Promega). The relative fold expression values were determined by the ΔΔCt method and normalized to GAPDH as a reference gene. Primers are available in Supplementary Table S1.

To know the expression levels of PU.1 in macrophages under normal conditions, PKM, LYC-FM, or LYC-RM cells were collected. Total RNA was extracted using Eastep Super Total RNA Extraction Kit (Promega), and first-strand cDNA was synthesized from the total RNA by using an Eastep RT Master Mix (Promega). Quantitative real-time PCR (qRT-PCR) was performed with the gene-specific primers (see Table S1). The reactions were conducted in duplicate and each 10 μl reaction volume contained 2 μl cDNA template, 5 μl SYBR qPCR master mix (Vazyme Biotech Co., Ltd.), and 0.25 μl of each forward and reverse primer (10 μM). The amplification profile was analyzed using Quant Studio 5 (Applied Biosystems). Gene expression levels were normalized to that of β-actin.