Direct zol rna miniprep

RNA from WB and PBMC was isolated using Zymo Research Direct-zol RNA mini-prep (Zymo Research) per manufacturer’s instructions. Concentration and integrity of RNA was determined using an Agilent 2100 Bioanalyzer. Ribosomal RNA (rRNA) was depleted using the ClontechRibo-Gone rRNA Removal kit. Libraries were constructed using the ClontechSMARTer Stranded RNA-Seq kit. First, rRNA-depleted RNA was fragmented and converted cDNA. Adapters were ligated and the ~300 base pair (bp) fragments were then amplified by PCR and selected by size exclusion. Each library was prepared with a unique indexed primer for multiplexing. Quantitation and quality of libraries were confirmed on the Agilent 2100 Bioanalyzer. Multiplexed libraries were subjected to single-end 100 bp sequencing using the Illumina HiSeq. 2500 platform. RNA-sequencing data presented in this article were submitted to the National Center for Biotechnology Information Sequence Read Archive (Accession number PRJNA398558).

RNA from each cell subset was isolated using Zymo Research Direct-zol RNA mini-prep (Zymo Research, Irvine, CA, USA) per manufacturer’s instructions. RNA concentration and integrity was determined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Ribosomal RNA (rRNA) was depleted using the ClontechRibo-Gone rRNA Removal kit. Libraries were constructed using the ClontechSMARTer Stranded RNA-Seq kit (Takara Bio Inc., Kusatsu, Shiga, Japan). First, rRNA-depleted RNA was fragmented and converted to double stranded cDNA. Adapters were ligated and the ~300 base pair long fragments were then amplified by PCR and selected by size exclusion. Each library was prepared with a unique indexed primer for multiplexing. In order to ensure proper sizing, quantitation, and quality prior to sequencing, libraries were analyzed on the Agilent 2100 Bioanalyzer. Multiplexed libraries were subjected to single-end 100 base pair sequencing using the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA).

Total RNA from cells or tissues were isolated using Direct-zol RNA Mini Preps (Zymo Research). Total RNA was reverse-transcribed and cDNA was quantified by qPCR with mRNA specific primers (Table 1). Relative expression levels of Adam proteases and Relt were calculated by the 2^−ΔΔCt method using Gapdh or 18s rRNA Ct values as the housekeeping gene control^{35 (link)} (*p < 0.05; **p < 0.01).Table 1

Primers Used for Real-Time quantitative PCR Results.

Gene name	Forward Primer	Reverse Primer
18s rRNA	GTAACCCGTTGAACCCCATT	CCATACCAATCGGTAGTAGCG
Adam8	CCACTCCCAGTTCCTGTTTATG	TTGACCTGCTTGGGTTTCAG
Adam9	GCCACCTGGGCATGGAAA	TATTTTCGGTTGTGGTGGAGGT
Adam10	ATGACTGGAGTAGAGGAAGGAG	TCTTTCAGCCAGAGTTGTGC
Adam15	CCCGCCGCTGCCAAATATAG	GATCCACTCAGCGTCCTCTC
Adam17	GGGATCTACAGTCTGCGACA	CCACCACCACGACTCTCAAG
Adam19	AGCTTTACCTGGTGGCTGATTA	TTCAGGGAGCGGTAAAACTTAT
Gapdh	ACTGGCATGGCCTTCCG	CAGGCGGCACGTCAGATC
Relt	CTGATGATGAAGCGGACCTTG	GGAGTTATTGGTGTTGGAGTGG

High-molecular-weight genomic DNA was isolated from ∼30 mg of tissue using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. DNA quality and integrity were assessed with a Nanodrop 2000 photometer (Thermo Fisher Scientific, Waltham, MA, USA) and through visual inspection on a 2% agarose gel. Exact quantity was determined using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). Total RNA was isolated by incubating 30 mg of tissue with 1 mL TRIzol (Invitrogen) in a 2 -mL tube for 1 hour. Further tissue disruption and homogenization was performed in three 1-minute cycles with grinding beads using a TissueLyser II (Qiagen). RNA was purified from the homogenized sample with Direct-Zol RNA minipreps (Zymo, Irvine, CA, USA) following the manufacturer's protocol. Quantity and quality were assessed using a RNA Pico chip on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA).

Extraction of RNA from samples in NZYol (NZYtech, MB18501) was achieved by using the Direct-zol RNA minipreps (Zymo Research, R2052). Reverse transcription (RT) was performed using the transcriptor first strand cDNA kit (Roche, 04896866001). Real-time RT-PCR to detect GAPDH and IFN- β, IFN-α, IL-29, and Viperin was prepared in 384-well, white, thin walled plates (Biorad, HSP3805) by using SYBR Green Supermix (Biorad, 172-5124), 10% (v/v) of cDNA and 0.4 µM of each primer. The reaction was performed on a CFX 384 Touch Real-Time PCR Detection System machine (Biorad), under the following PCR conditions: Cycle 1 (1 repeat): 95 °C for 2 min; Cycle 2 (40 repeats): 95 °C for 5 s and 60 °C for 30 s; Cycle 3: 95 °C for 5 s and melt curve 65 °C to 95 °C (increment 0.05 °C each 5 s). Data were analyzed using the CFX manager software (Biorad).
Primer sequences used for real-time RT-qPCR were the following:
GAPDH Fw: 5′-CTCTGCTCCTCCTGTTCGAC-3′;
GAPDH Rv: 5′-ACCAAATCCGTTGACTCCGAC-3′;
IL-29 Fw: 5′-AATTGGGACCTGAGGCTTCT-3′;
IL-29 Rv: 5′- GTGAAGGGGCTGGTCTAGGA-3′;
IFN-β Fw: 5′- CCTGAAGGCCAAGGAGTACA-3′;
IFN-β Rv: 5′- AAGCAATTGTCCAGTCCCAG-3′
IFN-α Fw: 5′- ATGGCCCTGTCCTTTTCTTT-3′
IFN-α Rv: 5′- ATTCTTCCCATTTGTGCCAG-3′
Viperin Fw: 5′- TCACTCGCCAGTGCAACTAC-3′
Viperin Rv: 5′- TGGCTCTCCACCTGAAAAGT-3′

Total RNA was extracted from TRIzol® preserved samples using Direct-zol RNA Minipreps (Zymo, Irvine, CA). RNA quantification was carried out by ND-1000 (NanoDrop) then converted to cDNA using 1ug of RNA using iScript™ cDNA synthesis kit (Biorad, Hercules, CA) per manufacturer’s specifications. For qRT-PCR analysis, 1 µl of cDNA per 10 µl reaction was amplified using iTaq™ Universal SYBR® Green Supermix (Biorad, Hercules, CA). Expression changes were calculated using ΔΔCt method with reference to Gapdh and relative expression was calculated and normalized to WT or sham control sample. All primers were tested for primer efficiency and used at 80–105%.

Expression levels of Wx during endosperm development were analyzed at 5, 10, 15, and 22 days after flowering (DAF). Because inflorescences of Setaria species have multiple orders of branching and flowers on the different branches open at different times, we recorded the day of anthesis as the beginning of flowering and to study endosperm development we sampled the same parts of the inflorescence where anthesis was first observed [44 (link)]. Total RNA was extracted using Direct-zol RNA MiniPreps (Zymo Research Corp, USA) and was applied to generate the first strand of cDNA with PrimerScript RT Reagent Kits (TaKaRa Bio Inc., Japan). The first strand cDNA was amplified in a 20 μl qRT-PCR reaction (KAPA SYBR FAST qPCR Kit Master Mix, KAPA Biosystems, USA). For each sample, three biological replicates of qRT-PCR reactions were performed with Elongation factor 1-alpha (EF1a) as the internal control. The newly designed primers were for both GBSSI (F: ctccacaccaccaccaaggg/ R: gcagctggttgtccttgtaa) and EF1a (F: cactcttggagtgaagcagatgatc/ R: cgaaggcaatcttgtcagggttgta) in qRT-PCR and RT-PCR. Significant differences in expression levels of Wx were determined by using two-way analysis of variance (ANOVA) and the Fisher’s Least Significant Difference (LSD) test at p < 0.05 level by R (version 3.4.2).