Superscript 3 and random hexamers

The rice cultivar Azucena and the Pfv-like strain IRRI 7007 were used in a time course gene expression experiment. At maximum tillering, the leaf sheath of rice was syringe-infiltrated with Pfv inoculum and sampling points 0, 3, 24, 48, and 72 hours post infection (hpi) were considered. IRRI 7007 was chosen because of its high virulence. For semi-quantitative RT-PCR, the total RNA from the treated rice sheath tissues of Azucena, as well as tissues from mock samples, were obtained using the Trizol method (Invitrogen, USA). Complementary DNA (cDNA) was synthesized from the pooled RNA molecules using SuperScript III and random hexamers (Invitrogen, USA). Primers were designed to target the coding sequences of candidate pathogenicity genes (S3 Table). In addition, two core random genes were selected. RT-PCR was done with the gene-specific primers in a 20ul reaction mix and was performed in a thermal cycler machine (G-storm GS1). We used the 16S gene to normalize the Pfv-like gene expression assessment. This experiment was done in two independent biological replicates with three technical replicates per sample. RT-PCR products were visualized on 1.25% agarose gels.

Cellular RNA was extracted with Trizol reagent (Invitrogen) and 1 ng was reverse transcribed with Superscript III and random hexamers (Invitrogen). Quantification of gene expression of the three PI5P4K isoforms was assessed by using TaqMan Gene Expression Assays and TaqMan Gene Expression Master Mix (Life Technologies), using TATA-box-binding protein (TBP) as the reference gene.

For each biological triplicate, 30 μg of total RNA was extracted from HEK293T cells transduced with lentivirus expressing WT or m⁶A-mutant reporters at a MOI of 3 and was fragmented to ∼100 nt fragments using 10x Ambion Fragmentation Reagent for 10 min at 75 °C. Fragments were immunoprecipitated with 5 μg of Rabbit anti-m⁶A antibody (Abcam) precoupled to 50 μl of Protein A/G magnetic beads (Pierce) in 300 μl of immunoprecipitation buffer (50 mM Tris–Cl pH 7.4, 150 mM NaCl, 1% NP-40 substitute, 0.5 mM EDTA) for 1 h at 4°C. Following three washes in IP buffer and in two washes in 0.25× SSPE, RNA was eluted twice with 125uL of elution buffer (50 mM Tris–Cl pH 7.4, 1 mM EDTA, 20 mM DTT, 0.1% SDS) by incubating at 42°C for 5 min. RNA was purified using acidic phenol (Sigma) and chloroform, and ethanol precipitated in the presence of 1 ul of GlycoBlue Coprecipitant (Ambion). RNA was finally resuspended in 10 μl of RNAse free water. cDNA was prepared with Superscript III and random hexamers (Invitrogen) and Real-Time PCR was performed using iTaq Universal SYBR Green Supermix in a CFX Connect Real-Time PCR system (Bio-Rad). To control for immunoprecipitation efficiency, the enrichment of WT and mutant reporters pulled down in each replicate was normalized to the enrichment of an endogenous m⁶A site found in ACTB.

Gonadal tissue from each embryo was processed and analyzed individually. Total RNA was extracted and subjected to DNase treatment using an RNeasy Micro kit (Qiagen) in accordance with the manufacturer's instructions, and quantified with a NanoDrop spectrophotometer (NanoDrop Technologies). cDNA was synthesized by reverse transcription with SuperScript III and random hexamers (Invitrogen), from 100 ng total RNA (for analyses of precisely-staged embryos between 8–30 ts, within a single transgenic line) or from 300 ng total RNA (for all other analyses, at 11.5 or 13.5 dpc). All qRT-PCR reactions were performed in triplicate using SYBR Green PCR master mix (Invitrogen) and 150 nM each of forward and reverse primers, and analyzed on a Viia7™ Real-Time PCR System (Invitrogen). Sry cDNA was amplified with primers 5′-TTATGGTGTGGTCCCGTGGT and 5′-GGCCTTTTTTCGGCTTCTGT [34] (link) and Sox9 cDNA was amplified with primers 5′-AGTACCCGCATCTGCACAAC and 5′-TACTTGTAATCGGGGTGGTCT. Relative cDNA levels were determined by calculating 2−ΔCt values relative to the house-keeping gene Sdha, using primers 5′-TGTTCAGTTCCACCCCACA and 5′-TCTCCACGACACCCTTCTGT. Sdha was verified previously as a suitable normalization gene for gonadal tissue [35] (link). qRT-PCR data is presented as the mean 2−ΔCt value for multiple individual embryos (minimum of 3). Sample sizes are indicated in relevant figures.