Nanodroptm 2000

The cells were cultured as described in Section 2.5. Total cellular RNA in each well was collected using Total RNA Kit I (Omega R6834-01, Omega, Guangzhou, China) following the manufacturer’s instructions and the concentration of acquired RNA was measured using a NanoDrop^TM 2000 spectrophotometer (ThermoFisher, Waltham, MA, USA). The RNA from each group was then reverse transcribed into complementary DNA (cDNA) using TransScript II All-in-one Fist-Strand cDNA Synthesis SuperMix. The cDNA was then amplified and analyzed by qRT-PCR (TransGen Biotech, Beijing, China) with TransStart Green qPCR SuperMix (TransGen Bioteh, Beijing, China) and primers. The relative expressions of osteoblastic differentiation-related genes, including ALP, osteopontin (OPN), RUNX family transcription factor 2 (RUNX2), collagen-I (COL-I), and osteocalcin (OCN) were quantified using the cycle threshold value and 2^−ΔΔCT method. The expression of housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control for normalization. PCR primers sequences are provided in the Table S1.

Total RNA was extracted by a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. RNA concentrations were quantified by NanoDropTM 2000 (Thermo Scientific, Waltham, MA, USA). Reverse transcription reaction was performed using 2μg of total RNA with Quantscript RT Kit (Tiangen biotechnology, Beijing, China). The mRNA expression level was determined by RT-qPCR using Bestar SybrGreen qPCR mastermix (DBI, Shanghai, China) and LightCycler 480^® II Real-Time PCR System (Roche, Basel, CH). Primers used are listed in Table 1.

Genomic RNA and total DNA were isolated from cell lines and tissues using TRI Reagent® (Molecular Research Center, Cincinnati, OH, USA) and DNAzol® reagent (Invitrogen, Rockville, MD), respectively, according to the manufacturer's instructions. The concentration of the samples was determined by spectrophotometry using a NanoDropTM 2000 (Thermo scientific). Reverse transcription of RNA was using GoScriptTM Reverse Transcription (Promega, Madison, WI, USA) 59 (link). Semi-quantitative RT-PCR was carried out with Go-Taq DNA polymerase (Promega, Madison, WI, 487 USA) under the conditions detailed in a previous study 59 (link), 60 (link). Real-time PCR (qRT-PCR) was performed with SYBR (Promega) on an HT7500 Real-Time PCR system (Applied Biosystems, Foster, CA, USA) according to the instrument manual 59 (link). The relative expression was estimated with the 2^-△Ct method 59 (link), and all assays were performed in triplicate. GAPDH or Actin was amplified as a loading control for RNA integrity. All primers and reaction systems are listed in Table 1.

A total of 66 samples were sequenced including (i) 64 population samples at 100 generation intervals from the eight independently evolved lineages and, (ii) two parental strains of E. coli MG1655 wild-type and ΔmutS mutator strain. Genomic DNA (gDNA) was isolated using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, United States) as per the manufacturer’s protocol. The isolated gDNA was checked using a NanoDrop^TM 2000 (ThermoFisher Scientific, Waltham, MA, United States) to assure UV absorbance ratio between 1.8 ∼ 2.0 and visualized under 1% agarose gel. The DNA concentration was quantified via a Qubit^® 2.0 Fluorometer (Invitrogen, Carlsbad, CA, United States) using a Qubit^TM dsDNA HS Assay Kit (Invitrogen, Carlsbad, CA, United States). For each sample, 2,200 ng input DNA was suspended in 56 μL of TE Buffer (pH 8.0) and sheared to target peak size of 400–500 bp using the Covaris S220 Focused-ultrasonicator (Covaris, Woburn, MA, United States) according to manufacturer’s recommendations, except that sonication duration was subjected to variation. NGS library was prepared using a TruSeq DNA PCR-Free kit (Illumina FC-121-3002) following the manufacturer’s guide. Resulting libraries were sequenced with rapid-run mode as 50 cycle single-ended reaction in the Illumina Hiseq 2500 system.

The samples were immediately transported to the laboratory in ice-cooled containers and stored at –70˚C until DNA extraction was performed. Each sample was thoroughly mixed using a spatula and divided into 250–300 mg aliquots. The total DNA was extracted using the Fast DNA SPIN Kit for Feces (MP Biomedicals, #116,570,200) following the manufacturer's instructions. DNA purity and concentration were evaluated by measuring the absorbances (ABS) at 260 and 280 nm using a NanoDropTM spectrophotomer (NanoDropTM 2000, Thermo Fisher Scientific Inc., Wilmington, DE, USA). All the DNA samples had ABS₂₆₀/ABS₂₈₀ ratios of 1.8–2.0. Illumina HiSeq4000 Platform (Illumina, San Diego, CA, USA) was used to sequence the DNA samples. We used the TruSeq DNA PCR Free Kit (Illumina, San Diego, CA, USA) and did not include a PCR amplification step.

The miRNA content from the aorta, the liver and the cells were extracted following the mirVana^TM miRNA Isolation Kit (Invitrogen^TM, Thermo Fisher Scientific, Waltham, MA, USA). The miRNA content from paraffin-embedded carotids was extracted using the RNeasy FFPE kit (Qiagen, Hilden, Germany). All the extractions were made following the protocol handled by the manufacturers. In all cases, miRNAs and long RNAs were obtained in separate fractions. The miRNA sample concentration was then determined using a NanoDrop^TM 2000 and the NanoDrop 2000/2000c Operating Software (Thermo Scientific, Waltham, MA, USA).

HepG2 cells were seeded in a 6-well plate. After incubation for 24 h, cells were treated with OTA for 48 h, after which RNA was isolated with 500 μL of RNAiso Plus (TaKaRa Co., Ltd., Kusatsu, Japan) per well. The amount of RNA isolated was confirmed by NanoDrop^TM2000 (Thermo Scientific, Rockford, IL, USA). cDNA was synthesized from RNA using a first-strand cDNA synthesis kit (LeGene Biosciences, San Diego, CA, USA).