Mx3005p system

Total RNA was extracted from the rice plants using a TRIzol reagent (Takara, Dalian, China). DNA removal and cDNA synthesis were performed using a PrimeScript RT reagent Kit (Takara, China), following the manufacturer’s instructions. Subsequently, a qRT-PCR assay was performed using Ssofast EvaGreen Supermix (BIO-RAD, Hercules, CA, USA) on an Mx3005P system (Agilent, Santa Clara, CA, USA) using rice ubiquitin (LOC_Os03g13170) as the internal reference gene [42 (link)]. Three technical replicates were maintained per treatment. The primers used for the qRT-PCR are listed in Table S1.

Total RNA from cell pellets was extracted using the Nucleospin RNA kit (Macherey-Nagel). Quantity and quality of RNA were evaluated by NanoDrop dosage and gel electrophoresis. RNA was reverse transcribed and conventional PCR was done with primers and PCR parameters described in Supplementary Tables S3 and S4 49 (link)50 (link). Primers for Real-time PCR were designed by the Primer3 online software (Supplementary Table S5). After mixing 2 μL of cDNA, primers and SYBR Green, high-throughput Real-Time PCR was realized on the BioMark HD System (Fluidigm). Results were visualized on the Real-Time PCR Analysis software (Fluidigm) and analyzed by the 2ΔΔCt method using the geometrical mean of Ct values for the three reference genes (ACTB, GAPDH and TBP) as a reference and the I3 cell line in LIF+2i medium as a calibrator. Statistical analysis was performed by t-test for two-by-two comparisons and 2-way ANOVA for multiple comparisons. Conventional real-time PCR was performed on an Agilent Mx3005p system. Results were visualized on the MxPro software (Agilent) and analyzed by the 2ΔΔCt method using the geometrical mean of Ct values for the reference genes RPL4 and HPRT as a reference49 (link).

RNA was isolated using TRIzol protocol (TRI Reagent, Sigma^® #T9424), in which Chloroform (Sigma^® #C2432) is as denaturalization agent for the cells and Isopropanol (Sigma^® #650447) is the RNA precipitating agent. The precipitate was diluted in DEPC water (Ambion^® #AM9920) containing 1% SUPERaseIn RNase Inhibitor (Thermo Fisher^® #AM2694). One μg of RNA was treated with DNase I (Invitrogen^® #18068015) prior to their reverse transcription by SuperScript^® III First-Strand Synthesis System kit (Invitrogen^®, #18080051). The cDNA obtained was employed for the gene expression analysis, which was performed using FastStart Universal SYBR GREEN Master (ROX) (Roche^® #04913914001) in a Mx3005P system (Agilent Technologies^®). The PCR amplification conditions were a denaturating step with 30 s at 95 °C; 45 cycles of 30 s at 95 °C, 1 min at 60 °C, and 30 s at 72 °C; followed by a cooling step. Each sample was run in triplicate in each experiment. Primers used: IL6 (forward, 5′-CCCAACAGACCTGTCTATACCA-3′; reverse, 5′-CAGAATTGCCATTGCACAAC-3′), TNFα (forward, 5′-CCACCACGCTCTTCTGTCTA -3′; reverse, 5′- AGGGTCTGGGCCATAGAACT-3′) and HPRT as housekeeping (forward, 5′-CAATGCAAACTTTGCTTTCCC -3′; reverse, 5′-TCCTTTTCACCAGCAAGCTTG -3′).

Real Time PCR (qPCR) was used to validate CNAs of CCNL1 and PIK3CB genes with the internal control HBB gene using SYBR-Green II fluorescence and Mx3005P System (Agilent Technologies, CA, USA)) (Supplementary Table S4). The results were analysed using the MxPro QPCR software. Comparative CT method was used to calculate the copy numbers of target genes. Cases with 2^−ΔΔCT > 1 were considered as amplification of genes, and cases with 2^−ΔΔCT < 1 were considered as deletion of genes [49 (link)].

RNA samples were used for quantitative reverse transcription-PCR (qRT-PCR) to quantify relative expression levels using a Stratagene MX3005P system, a Brilliant II SYBR green qRT-PCR master mix kit (Agilent), and primers specific to mtlA, mtlS, and 4.5S RNA. The reaction mixtures were set up in 96-well optical reaction plates and contained 1× Brilliant SYBR green qPCR master mix, 30 nM carboxy-X-rhodamine reference dye, each primer at 100 nM, 100 ng RNA, and 1 μl reverse transcriptase-RNase block enzyme mixture in a 25-μl reaction mixture. The following conditions were used for cDNA synthesis and PCR: 30 min at 50°C, 10 min at 95°C, and 40 cycles of 30 s at 95°C and 1 min at 60°C (Agilent). MxPro QPCR software (version 4.10) was used to determine the threshold cycle (C_T) values for each reaction, and relative RNA concentrations were calculated from the C_T values by comparison to standard curves. All transcript levels were normalized to a 4.5S RNA endogenous control. No signals were detected in the no-template controls and no-reverse transcriptase controls. Statistical analysis was performed using GraphPad Prism (version 7) software.