Rq1 dnase 1

Three important stages of N. caudatum fruits, early cotyledon development stage (52 DAF), fast oil accumulating stage (96 DAF) and fully maturation stage (146 DAF) from three individuals, were selected for RNA-seq analysis [2 ]. Total RNA of the fruits was extracted by using the RNeasy Plant Mini Kit (Qiagen, USA). DNase I (RQ1, Promega, USA) was added to remove any genomic DNA contamination. Total RNA was quantified using Nanodrop ND-2000 spectrophotometer (Nanodrop Technologies, USA). All the samples showed a 260/280 nm ratio of 1.8 to 2.1. The poly-A tailed mRNA was purified from the total RNA using Dynabeads™ mRNA Purification Kit (Cat # 61006,Thermo Fisher Scientific, USA). The first-strand cDNA fragments were synthesized with random primers and transformed into double-strand cDNA. Fragments of desirable lengths (200-300 bp) were purified, end-repaired and ligated with the sequencing adapters through A and T complementary base pairing. The sequencing library was constructed using polymerase chain reaction (PCR). The synthesized cDNA libraries were normalized to a 10 nM and gradually diluted and quantified to 4-5 pM.

Transcription of the ces gene cesB was analyzed by qRT-PCR as described previously (Dommel et al., 2011 (link)). In brief, RNA was isolated from frozen cell pellets via TRIzol Reagent (Invitrogen) and bead beating. Phase separation was carried out with chloroform and nucleic acid was precipitated with 75% ethanol. Subsequently, DNA was digested with RQ1 DNAse I (Promega) and a total amount of 1 μg RNA was used for cDNA synthesis (cDNA qScript Supermix, Quanta Biosciences). qRT-PCR for each sample was performed in triplicates on a C1000 Touch Thermal Cycler CFX96 Real-Time System (BioRad). In order to normalize and compare the relative expression the REST method was employed (Pfaffl et al., 2002 (link)), which has been shown previously to be a quite suitable tool for analysis of ces transcription (see, e.g., Lücking et al., 2009 (link), 2015 (link); Dommel et al., 2010 (link), 2011 (link); Frenzel et al., 2012 (link)). As internal calibrator, (with a relative expression value of 1.00) ces gene expression at an OD₆₀₀ of 1.0 was chosen since it has been shown previously to be consistent throughout experiments (Dommel et al., 2011 (link)). Samples at OD₆₀₀ of 1 and 10 were always taken from the same cultures. Sample to sample variation was corrected by using the 16S rDNA gene as a reference (rrn). Mean values and standard deviations were calculated from two independent experiments.

PER2::LUC organoids were plated into 12-well plates at the start of the experiment. Organoids for each time point were plated into a separate plate to limit manipulation or exposure to possible resetting cues. End of serum shock is indicated by circadian time 0 to compare with bioluminescent recordings performed in parallel. RNA was harvested every 4 hours over a 48-hour period using 0.25 ml/well of TRI Reagent™ RT (Molecular Research Center, Cincinnati, OH, USA). Suspensions were passed through a syringe needle several times to break up the Matrigel™ and organoids. Samples were immediately stored at −80°C until all time points were collected and could be processed. Total RNA was treated with RQ1 DNaseI (Promega, Fitchburg, WI, USA). For reverse transcription reactions, 1 μg of total RNA was used with GoScript Reverse Transcriptase (Promega) according to manufacturer’s instructions. qPCR was performed with SYBR Green Master Mix (Qiagen, Germantown, MD, USA). qPCR results were detected by using the StepOnePlus System (Life Technologies, Grand Island, NY, USA).

Total RNA was purified according to TRI Reagent standard procedure (Sigma, USA), following digestion with RQ1 DNase I (Promega, USA) to remove contaminating genomic DNA. Afterward, 500 ng of DNA-free RNA was reverse transcribed using Omniscript RT kit (Qiagen, Germany) with oligo-dT primer. The RT reaction was performed at 42°C for 60 min. PCR mixtures contained 1 μL cDNA, 1 μL of each primer (10 μM), and 12.5 μL 2x Taq MasterMix (Qiagen, Germany) in a final volume of 25 μL. Specific sense and antisense primers, annealing temperatures, and number of repeating cycles for both studies are referred to in Table 1. Amplifying conditions were performed as follows: a first step of 95°C for 5 minutes, thereafter repeating cycles comprised of 95°C for 30 seconds, specific annealing temperature for 30 seconds and 72°C for 30 seconds, and a final extension step of 5 minutes at 72°C. PCR bands (8 μL of PCR product plus 2 μL of gel loading buffer) were resolved on 1.5% (w/v) agarose gel electrophoresis and visualized under ultraviolet light subsequent to being stained with ethidium bromide. PCR products were quantified using the Kodak ID 3.6 software package (Kodak Inc, USA). Beta-2 microglobulin was used as housekeeping gene for normalization.

Total RNA concentration and quality were determined by UV spectrometry (NanoDrop ND1000 spectrophotometer; Thermo Fisher Scientific), and all samples were brought to a final concentration of 150 ng/μl. Total RNA (1.5 μg) was treated with RQ1 DNase I (M610; Promega) and subsequently assessed by conventional PCR for the complete absence of genomic DNA (gDNA). Subsequently, 1 μg of DNase-treated RNA was reversed transcribed using SuperScript II reverse transcriptase (18064-014; Invitrogen, Thermo Fisher Scientific), with random hexamers (11034731001; Roche Diagnostics) as primers. Prior to qPCRs, cDNA samples were diluted 1:3 in water, except in the case of reactions against the 18S rRNA, for which samples were diluted 1:1,200 in water. qPCRs were performed on a Bio-Rad CFX96 instrument. A typical qPCR mixture (10 μl) contained 13.5 ng of cDNA (32 pg when the qPCR was for 18S rRNA), a specific pair of primers (150 nM final concentration), and the SYBR premix Ex Taq (RR420A; TaKaRa). Primers used for qPCR are listed in Table 5. For all qPCRs, 40 cycles were performed using the following cycling conditions: denaturation at 95°C for 10 s, annealing at 59°C for 20 s, and extension at 72°C for 20 s. The relative expression ratio (rER) was calculated using the threshold cycle (2^−ΔΔCT) method (42 (link)) using the expression level of the 18S rRNA as an internal reference.