Gene specific taqman probe

Gingival tissues were dissected out from the rats 3, 7 and 14 days post-wounding, and HGFs were cultured on collagen type I and rhPN pre-coated plates as previously described for 1 day and 7 days. Total RNA was isolated using 1 ml of TRIzol® reagent (Ambion; Carlsbad, CA, USA) per well according to the manufacturer’s recommendations. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed on 50 ng of total RNA using TaqMan qScript^TM One-Step qRT-PCR Kit (Quanta; Gaithersburg, MD, USA) and gene-specific TaqMan probes (Applied Biosystems; Carlsbad, CA, USA) under following conditions: 48 °C for 30 minutes followed by 90 °C for 10 minutes and 40 cycles of 95 °C for 9 seconds and 60 °C for 1 minute using 7900 Real Time PCR system (Applied Biosystems). Postn, Acta2, Col1A2, Col3A1, Fn1, Lox, POSTN, ACTA2, COL1A2, COL3A1, and FN1 mRNA expressions were normalized to the housekeeping gene, 18 S. PCR efficiency was verified by dilution series and relative mRNA levels were calculated using the ΔΔCT method^{66 (link)}.

Total RNA was extracted from frozen powderized BAT, scWAT and spleen using Qiazol (Qiagen) and the RNeasy Mini Kit (Qiagen), performing on-column DNAse digest. cDNA was prepared by reverse transcription of 1 μg of total RNA using the First strand cDNA synthesis kit (Fermentas). Real-time RT-PCR was performed in order to measure gene expression of browning (Ucp1, Cidea, Dio2, Pparg, Prdm16) and inflammatory (Cd68, Ccl2, Tnfa, Ifng, Mrc1, Mgl1, Arg1, Il-10, Il-4) markers in BAT and scWAT and to verify T_reg cells depletion from BAT and spleen by measuring FoxP3 expression. TaqMan Master Mix (Applied Biosystems) and a gene specific Taqman probes (Applied Biosystems) were used on an ABI StepOnePlus sequence detector (Applied Biosystems). Relative mRNA expression levels were calculated by the delta Ct method using TATA-box binding protein (Tbp) expression as a reference.

AD-MSC-derived exosomes (10 μg/ml) was used to treat JEG-3, and HTR-8 cells for 24 h. Then, we isolated the total RNA from the trophoblasts with TRIzol Reagent (Takara, Japan). After that, a reverse transcription kit (Invitrogen) was used to synthesize complementary DNA. Master Mix (Thermo Fisher Scientific) and Gene-specific TaqMan probes (Applied Biosystems) were used to carry out quantitative real-time PCR (RT-PCR) according to the manufacturer’s instructions. The expression of each target gene was normalized to GAPDH expression. We used TaqMan probes for EZH2 (Hs00544830_m1), mTOR (Hs00234508_m1), S6K1 (Hs00356367_m1), and GAPDH (Hs02786624_g1), and conducted three separate reactions for each marker.

Dissected tissues were immediately frozen in liquid nitrogen, homogenized using Precellys24 (Precellys, Bertin Technologies, Paris, France) into RPL buffer (Qiagen) and the RNA was extracted with RNeasy Mini Kit (Qiagen). cDNA was produced with SuperScriptII (Life Technologies) and 50ng of cDNA was used for RT-Q-PCR with TaqMan Universal MasterMix II and gene-specific TaqMan probes (Life technologies, Supplementary Table 2). The fold change related to Actin-b was calculated using 2^-ΔΔTh method (Livak & Schmittgen 2001 (link)).

Metabolic labeling of cells for purification of nascent transcript was carried out as described (Rädle et al., 2013 ). Cells were treated with tamoxifen for the times indicated and pulse labeled with 500 μM 4-thiouridine for 15 minutes prior to harvesting in Trizol (Life Technologies) for RNA purification. For isolation of total RNA for sequencing, cells were harvested in Trizol and processed according to manufacturer’s instructions.
Libraries for sequencing were prepared using the NEXTflex Rapid Directional RNA-seq kit (Illumina) or SMARTer Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (Takara Bio) and sequenced as above. Individual expression assays were carried out using gene-specific TaqMan probes (Life Technologies).
RNA-seq libraries were mapped against the mouse reference genome mm10 using GSNAP (gmap-2014- 12-17) (Wu and Nacu, 2010 (link)) with parameters “–m 7 –i 1 –N 1 –w 100000 –E 100 –n 10.” Gene read counts were calculated with HTSeq (v0.6.1) based on gene annotation from Ensembl release 75, and normalization and differential expression analysis were performed using the R package DESeq2 (Love et al., 2014 (link)) with the default model. We identified differentially expressed genes at FDR-adjusted p-values less than 0.05. For time series analyses differential expression was assessed for all pairwise comparisons against the 0 h time point.