Superscript system

Total RNA was isolated from NP cells using TRIzol reagent (Invitrogen, Waltham, MA, USA). First-strand cDNA was synthesized using the SuperScript system (Invitrogen, Waltham, MA, USA). PCR amplification conditions were as follows: denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, and extension at 72 °C for all genes. PCR reactions were performed using the QuantiTech SYBR Green PCR kit (Qiagen, Valencia, CA, USA) and an Exicycler 96 system (Bioneer, Daejeon, Korea). Specific primers for the genes examined were based on their PrimerBank sequences and are listed in Table 1.

Double-stranded cDNA was synthesized using the Superscript system (Invitrogen), purified by phenol : chloroform extraction, and concentrated by ethanol precipitation. In vitro transcription was used to produce biotin-labeled cRNA (GeneChip In Vitro Transcription Labeling Kit, Affymetrix).

Total RNA was isolated from yeast cells using TRIzol (Invitrogen), and further purified with the kit RNeasy (Qiagen), following the manufacture's instruction. The cDNA was synthesized using the SuperScript system (Invitrogen). Following synthesis, cDNA purification was conducted after RNA degradation using adsorption chromatography, as described (Trujillo-Esquivel et al., 2016 (link)). Absence of genomic DNA in the cDNA preparations was confirmed by amplification of the ACT1 gene which contains an intron of 658 bp (data not shown).
All primer pairs used in qPCR reactions are listed in Table S4. The reaction mixtures were prepared using the SYBR® Green PCR Master Mix (Thermo Fisher Scientific) and analyzed in a StepOnePlus Real-Time PCR System (Applied Biosystems). All reactions produced a single amplicon, with a uniform melting curve, as determined by the dissociation profile of the products. Relative quantification was determined by calculating 2^−ΔΔCT (Livak and Schmittgen, 2001 (link)). Expression data were normalized using RPP2B, a housekeeping gene previously used as a control in expression assays (Nailis et al., 2006 (link)).

Total RNA was prepared from the murine CH27 cell line by RNeasy Maxi (QIAGEN Inc.) and was purified with FastTrack MAG Maxi mRNA isolation kit (Invitrogen) to obtain poly(A)+ RNA. cDNA was synthesized with the SuperScript system (Invitrogen) and was cloned into SPORT6 vector with SalI and NotI restriction sites. ElectroMAX DH10B competent cells (Invitrogen) were transformed by electroporation, and after titration, E. coli (~150 clones/well) were inoculated overnight into 96-well format culture blocks (10 blocks). Plasmids were purified with a Qiaprep 96 Turbo miniprep kit (QIAGEN) and were transfected to HEK293T cells with Lipofectamine 2000 (Invitrogen) in 96-well flat-bottom plates and left overnight. Hybridomas were cocultured with cDNA-transfected 293T cells for 24 h, after which mIL-2 amounts in the supernatants were obtained. Positive clones were selected for secondary and tertiary screenings. Subpool libraries (~20 clones/well, 48 wells) and clone libraries (1 clone/well, 96 wells) were prepared and screened. Positive clones were sequenced to identify the specificity of the transfected cDNA.

The quality of RNA was determined by using RNA 6000 Nano LabChip Kit and Agilent Bioanalyzer 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The total RNA from individual animal was further purified by using RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). Total RNA (3 μg) was converted to double-stranded cDNA using SuperScript System (Invitrogen, Carlsbad, CA) and an oligo (dT24) primer containing a T7 RNA polymerase promoter site. Biotin-labeled cRNA was synthesized from cDNA using a labeling kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by product absorbance at 260 nm measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), and a size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). Arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidic Station 400 (Affymetrix), according to the standard protocol of the manufacturer. The arrays were scanned by using GeneChip Scanner 3000 (Affymetrix).

The quality of RNA was determined by using an RNA 6000 Nano LabChip Kit and Agilent Bioanalyser 2100 (Agilent, Palo Alto, CA, USA). Preparation of cRNA was performed according to the protocol provided by Affymetrix (Santa Clara, CA). The same amounts of total RNA from two animals were pooled and further purified by using an RNeasy Mini Kit (Qiagen Inc. Total RNA (3 μg) derived from each pool (n = 4) was converted to double-stranded cDNA using a SuperScript System (Invitrogen, Carlsbad, CA) and an oligo(dT)24 primer containing a T7 RNA polymerase promoter site. Biotin-labelled cRNA was synthesised from cDNA using a labelling Kit and purified by using a GeneChip Cleanup Sample Module (Qiagen Inc., Valencia, CA, USA). The yield of the in vitro transcription reaction was determined by the product absorbance at 260 nm, as measured by NanoDrop ND-1000 (NanoDrop Technologies, Inc., Montchanin, DE), size of cRNA probes was evaluated by using RNA 6000 Nano LabChip Kit (Agilent, Palo Alto, CA, USA). Fragmented cRNA was used for hybridization to GeneChip® Rat Gene 2.0 ST arrays (Affymetrix). The arrays were washed and stained with streptavidin-phycoerythrin (Merck, Darmstadt, Germany) in Fluidics Station 400 (Affymetrix) according to the standard protocol of the manufacturer. The arrays were scanned by using a GeneChip Scanner 3000 (Affymetrix).

Total RNA was extracted using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, CA, USA), and reverse transcription using the SuperScript system (Invitrogen) and oligo(dT) primer was carried out according to the manufacturer’s instructions. Real-time PCR was performed to measure gene expression was carried out as previously described (Jaffé et al., 2012 (link); Nazri et al., 2017 (link)) using SYBR Green (Finnzymes) and an Opticon DNA Engine Continuous Fluorescence Detector (GRI Ltd.). PCR was performed using specific forward and reverse primers for each gene (Supporting information Table S1) at 95 °C for 10 min followed by 35 cycles of 95 °C for 15 s and 60 °C for 1 min. All gene expression analysis was performed with at least three independent biological replicates and the reactions were set up in triplicate for each sample. All data were standardized by normalizing to SAND or Yellow-Leaf-Specific gene8 (YLS8) expression (Remans et al., 2008 (link); Jaffé et al., 2012 (link)) and analysed using Opticon Monitor III software (Biorad). Quantification of the relative transcript levels was performed using the comparative Ct (threshold cycle) method.