Abi prism 7700 sequence detection

Total RNA was extracted from culture cells using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). Total RNA (5 μg) was reverse transcribed into cDNA with the SuperScript III First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA). The primer sequences were as follows: gastrin, 5′GAC GAG ATG CAG CGA CTA TGT 3′ (sense) and 5′GGG TCT GCC ACG AGG TGT 3′ (antisense) and β-actin, 5′CGG GAA ATC GT GCG TGA CAT T 3′ (sense) and 5′CTA GAA GCA TTT GCG GTG GAC 3′ (antisense). Real-time quantitative PCR was performed using ABI PRISM7700 Sequence Detection (Applied Biosystems, Foster City, CA, USA). β-Actin was used as an internal control. PCR reaction procedure was 28 cycles of 94°C denaturation for 30s, 62°C annealing for 30 s, and 72°C elongation for 30 s and a final elongation at 72°C for 10 min. The relative mRNA expression levels of gastrin were calculated using the 2^-△△Ct method.

The methylation levels of the selected differentially methylated genes, namely regulating synaptic membrane exocytosis 2 (Rims2), harvey rat sarcoma virus oncogene (Hras1), thymoma viral proto-oncogene 1 (Akt1), and kirsten rat sarcoma virus oncogene homolog (Kras), were determined by BSP. Briefly, 1 microgram of DNA was treated with the EZ DNA Methylation Kit (Zymo Research, Hiss Diagnostics, Germany). The modified DNA was then amplified by PCR with primers designed with the Methyl Primer Express software version 1.0 (Applied Biosystems, Foster City, CA, USA, S3 Table), which corresponded to regions on the microarrays. PCR products were purified using the MinElute Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) and cloned into the pMD18-T Vector (Takara). The plasmids were purified using the PureLink Miniprep Kit (Invitrogen, Thermo Scientific Inc, Waltham, MA, USA). The positive clones were confirmed by PCR, and no fewer than 10 clones were randomly selected for each mouse for sequencing using an automatic sequencer (ABI Prism 7700 Sequence Detection, Applied Biosystems, Foster City, CA, USA). Sequencing results were analyzed using QUMA (http://quma.cdb.riken.jp/top/quma_main.html) [23 (link)].

The total RNAs were extracted from Kupffer cells using the Miniprep Purification Kit (GeneMark). Real-time polymerase chain reaction was performed with the SYBR Green PCR Master Mix and ABI PRISM 7700 Sequence Detection Systems (Applied Biosystems, Foster City, CA) according to the manufactu rer’s suggested protocol. Sets of TNF-α, IL-6, and IL-1β primers were designed according to those genes documented in GenBank [22 (link)].

Samples from two biological experiments were collected and pooled together using TRIzol Reagent (Life Technologies, catalog number: 15596-018), and RNA extraction was performed using phenol–chloroform extraction method. For each sample, 1 μg of RNA was reverse transcribed to complementary DNA (cDNA) using iScript cDNA Synthesis Kit (Bio-Rad, catalog number: 17-8891) according to the manufacturer’s instructions. The synthesized cDNA was analyzed via quantitative real-time polymerase chain reaction (qRT-PCR) for osteogenic markers, such as OCN, runt-related transcription factor 2 (RUNX2), and secreted phosphoprotein 1 (SPP1) as well as additional genes, including solute carrier family 20 (phosphate transporter), member 1 (SLC20a1), and Nanog homeobox (NANOG). Primer sequences for each analyzed gene are provided in Table S1 in Supplementary Material. Reactions were performed using SYBR Select Master Mix (Life Technologies, catalog number: 4472908) and ABI Prism 7700 Sequence Detection (Applied Biosystems). Fold expression values were determined by $2^{-\mathrm{\Delta \Delta }{\mathrm{C}}_{\text{t}}}$ after normalizing each target gene with respect to the housekeeping gene (GAPDH) within the sample, and compared to undifferentiated hESCs as the control that was expressed as 1.

Bisulfite modification was performed with the EZ DNA Methylation Kit (Zymo Research, Hiss Diagnostics, Germany). The converted DNA was then amplified by PCR with primers detailed in Table 1. Primers were designed using Methyl Primer Express Software version 1.0 (Applied Biosystems, Foster City, CA, USA). PCR products were purified using agarose gels (Invitrogen, Carlsbad, CA, USA) and ligated to the pMD18-T Vector (Takara, Shiga, Japan). The plasmids were then purified using the PureLink Miniprep kit (Invitrogen, Thermo Scientific Inc., Waltham, MA, USA). Positive clones were confirmed by PCR, and a minimum of 10 clones from each mouse (n = 8 mice/group) were sequenced using ABI PRISM 7700 Sequence Detection (Applied Biosystems, Foster City, CA, USA). Sequence analysis was performed using QUMA [22 (link)].