Rt master mix

Total RNA was extracted using the RNeasy Plus Kit (Qiagen) according to the manufacturer’s protocol. Complementary DNA was then generated using the 5X RT MasterMix (ABM). Gene expression to validate RNA-seq analysis was quantified by quantitative reverse transcriptase–polymerase chain reaction using BrightGreen 2X qPCR MasterMix (ABM) on a QuantStudio 3 reverse-transcriptase PCR system (Thermo Fisher Scientific). Fold change in gene expression compared to endogenous controls was calculated using the ddCT method. Primer sequences are indicated in Supplementary Table 1 [28 (link)].

Total RNA was isolated with total RNA extraction reagent (Vazyme Biotech Co., Ltd.; cat. no. R401-01). According to the manufacturer's instructions, cDNA was synthesized using RT MasterMix (Applied Biological Materials, Inc.; cat. no. G485). The following conditions were used for RT: 25°C for 10 min, followed by 42°C for 15 min. qPCR reactions (10 µl) consisted of 5 µl PrimeScipt RT master mix (Takara Bio, Inc.), 0.5 µl primer (final concentration, 10 nM), 2 µl DEPC water and 2 µl cDNA. Reactions were run in a LightCycler 480 instrument II (Roche Diagnostics Co., Ltd.). qPCR with SYBR Green detection (Takara Bio, Inc.) was performed. qPCR amplification conditions were as follows: Initial denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec, 56°C for 30 sec and 72°C for 30 sec. The following primers were used for cDNA amplification: Lats2: Forward, 5′-GACGATGTTTCCAACTGTCGCTGTG-3′ and reverse, 5′-CAACCAGCATCTCAAAGAGAATCACAC-3′; matrix metalloproteinase (MMP)-2: Forward, 5′-CAAGTTCCCCGGCGATGTC-3′ and reverse, 5′-TTCTGGTCAAGGTCACCTGTC-3′; and GAPDH: Forward, 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-UUCUCCGAACGUGUCACGUTT-3′. Relative levels were quantified with the 2^−ΔΔCq method and were normalized to GAPDH (23 (link)).