Turbo dna free dnase 1

Callus material was collected and snap-frozen in liquid nitrogen for gene expression analysis. For each experiment, at least two independent biological replicates were used. Total RNA was extracted using the Spectrum Plant Total RNA Kit (Sigma-Aldrich). Total RNAs were digested with Turbo DNA-free DNAse I (Ambion) according to the manufacturer’s protocol. RNA concentrations were measured with a NanoDrop ND-1000 UV−Vis spectrophotometer (NanoDrop Technologies). One microgram of total RNA was reverse transcribed (RT) using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s instructions. PCR reactions were performed in optical 96-well plates in the StepOne Plus Real Time PCR System (Applied Biosystems). Five microliters of a 1:10 dilution of cDNA was amplified in 20 µL of reaction mix. The following thermal profile was used: 95 °C for 10 min, 40 cycles of 95 °C for 15 s, 60 °C for 30 s. Amplicon melting curves, were recorded after cycle 40 by heating from 60 to 95 °C with a ramp speed of 1 °C min⁻¹. Expression levels of each gene, relative to EIF4A, were determined using a modification of the Pfaffl method^{33 (link)}. Technical triplicates were performed for each biological sample. Primers used for RT-quantitative (q)PCR analysis are shown in Supplementary Table 1.

Total RNA from siliques at 2 and 5 DPA was extracted using the Spectrum Plant Total RNA Kit (Sigma). Total RNAs were digested with Turbo DNA-free DNase I (Ambion) according to the manufacturer’s instructions. The mRNAs were reverse transcribed using the SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the manufacturer’s protocol. PCR reactions were performed in an optical 384-well plate in the QuantStudio™ 6 Flex Real-Time PCR System (ThermoFisher Scientific), using FastStart Universal SYBR Green Master (Rox) (Roche), in a final volume of 10 µL, according to the manufacturer’s instructions. The following standard thermal profile was used for all PCR reactions: 95 °C for 10 min, 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. The data were analyzed using the QuantStudio Real-Time PCR Software v1.3 (Applied Biosystems). As a reference, a geometric mean between two house-keeping genes, EIF4A1 and AP2M, was used to normalize ELA1 expression. For each couple of primers, PCR efficiency (E) was estimated from the data obtained from standard curve amplification using the equation E = 10^–1/slope. Expression levels are presented as E^–ΔCt, where ΔCt = Ct_GOI – Ct_REF. The sequence of the primers used for qPCR can be found in Supplementary Table 7.

RNA was extracted from ~100 mg ground tissue using the E.Z.N.A Plant RNA kit (Omega Bio-tek, Norcross, USA) and genomic DNA removed using Turbo DNAfree DNAse I (Ambion, Foster City, USA). RNA quantity and integrity was verified using an Agilent BioAnalyzer 2100 with RNA Nano 6000 kit (Agilent Technologies, Santa Clara, USA) prior to RNA-seq. Construction of cDNA libraries and RNA-seq was performed by the National Genomics Infrastructure (NGI; Uppsala, Sweden) after ribosomal RNA removal using Illumina Ribo-Zero rRNA removal kit (Plant Leaf). Single-end RNA-seq was performed on a HiSeq 2500 High Output V4 platform (Illumina, San Diego, USA), generating 13–32 million reads.