Sybr select master mix

Total RNA was isolated from either MEF or embryos using a commercial kit, Trizol, (ThermoFisher, Cat. No. 10296028). The total RNA was then reverse-transcribed using the M-MuLV kit (New England Biolab, Cat. No. M0253L), and the subsequent cDNA was used as a template for quantitative PCR. The qRT-PCR analysis was performed with SYBR Select Master Mix (Bio-Rad) using the ViiA™ 7 Real-Time PCR System (Life Technologies). All qRT-PCR reactions were carried out for 40 cycles under standard PCR conditions with internal controls (Gapdh and β-actin). The results derived from qRT-PCR were further analyzed using the threshold (Ct) value. The ΔCt value was initially calculated by subtracting Ct value of a testing replicate of a given gene from the average Ct value of the internal control (Gapdh). The fold difference for each replicate was then calculated by raising the ΔΔCt value as a power of 2. The average and standard deviation for each sample were then calculated by compiling the normalized values. The information regarding the sequences and PCR conditions for oligonucleotides used for the current study is available as S8 File.

Total RNA from primary cells and lung tissue was prepared using RNeasy kit (Qiagen Sciences, Valencia, CA) as described previously (Kasam et al, 2019). Real‐time PCR was performed using the CFX384 Touch Real‐Time PCR detection system and SYBR select master mix (Bio‐Rad, Hercules, CA). Target gene transcripts in each sample were normalized to mouse hypoxanthine guanine phosphoribosyltransferase (Hprt) or human beta‐actin or Gapdh. All the real‐time primer details are provided in Appendix Tables S2 and S3.

ChIP‐seq PCR was performed using the ChIP assay kit (Cell signaling technology, Denver, CO) as described previously (Sontake et al, 2018). Briefly, lung‐resident fibroblasts were purified from lung stromal culture of CCSP/TGFα mice on Dox‐containing food for 8 weeks. Cells were cross‐linked with 1% formaldehyde, and immunoprecipitation was performed with anti‐WT1 (catalog 12609‐1‐AP, Proteintech, Rosemont, IL) or isotype control IgG (Cell Signaling Technology, Denver, CO) at 4°C as described in the manufacturer's protocol. DNA was purified, and ChIP‐qPCR was performed using the CFX384 Touch Real‐Time PCR detection system and SYBR select master mix (Bio‐Rad, Hercules, and CA).

Total RNA of each cell line was extracted using TRIzol reagent (Invitrogen) and was subjected to cDNA microarray. Microarray experiments were performed using the commercial service that was provided by Ebiogen, Inc. (Seoul, Korea). Isolated RNA was used to synthesise cDNA and amplified cRNA for hybridisation to the Agilent Human Gene Expression 4X 44K Microarray (Agilent Technologies, Palo Alto, CA, USA) according to the manufacturer’s instruction. Gene expression was quantified from the hybridisation images using Agilent Feature Extraction software 10.7 (Agilent Technologies, USA). Data normalisation and the selection of fold-changed genes were performed using GeneSpringGX 7.3.1 (Agilent Technologies). The functional annotation of the genes was performed according to the Gene Ontology Consortium (http://www.geneontology.org) and the QuickGO Database (https://www.ebi.ac.uk/QuickGO/).^{28 (link)}For RT-PCR experiment, total RNA was reverse-transcribed into cDNA using the two-step RT-PCR kit (Invitrogen). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed by using the SYBR Select Master Mix (Bio-Rad). The relative quantitative value was expressed by the 2^−ΔCt method using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as control. Each experiment was performed in triplicate and repeated more than twice.

Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. First strand cDNA was synthesized with Takara RNA PCR kit (Baoshengwu, Dalian, China) according to the manufacturer’s instructions. Gene expression levels were measured by real-time RT-PCR using SYBR Select Master Mix (BIO-RAD, Hercules, USA), on a CFX 96^TM Connect Real-Time system (BIO-RAD, Hercules, USA). Primers were designed with the DNAMAN software and synthesized by Generay Biotech Co., Ltd. (Shanghai, China). β-actin was used for normalization. Primer sequences are listed in Table 1.