Tri reagent

The retina was dissected out of the eye and immediately placed in TRI reagent on ice (T9424, Sigma-Aldrich). RNA was extracted from mouse retinas using TRI reagent following the manufacturer’s protocol (https://www.sigmaaldrich.com/US/en/technical-documents/protocol/protein-biology/protein-lysis-and-extraction/tri-reagent). RNA was converted to complementary DNA using the qScript complementary DNA synthesis kit (PSF-95047-100, Quanta bio) following the protocol provided by the company (https://www.quantabio.com/product/qscript-cdna-synthesis-kit/). Reverse Transcriptase-Polymerase chain reaction (RT-PCR) was performed with 50 ng of complementary DNA to amplify exons 3 to 5 (Table S1, available at https://www.ophthalmologyscience.org).

After surgical excision, tissue samples for nucleic acid extraction were immediately sliced into aliquots of about 100 mg, snap-frozen in liquid nitrogen, and stored at −80°C until use. Aliquots for DNA extraction were homogenized in 2 ml of chilled NaCl 0.9% w/v; cell lysis was achieved by using Igepal CA-630 (Sigma-Aldrich, St. Louis, MO, USA) 0.1% and lysis solution (NaCl 100 mM, EDTA 25 mM, SDS 1.6%, pH 8). Samples were treated with proteinase K/RNase, and DNA was extracted with a standard phenol/chloroform procedure.
For RNA extraction, each liver tissue aliquot (100 mg) was kept on ice, immediately homogenized in 2 ml of TRIReagent® (Sigma-Aldrich, St. Louis, MO, USA) and the homogenate stored at −80°C until use. RNA was extracted by guanidinium thiocyanate-phenol-chloroform-based method using TRIReagent® (Sigma-Aldrich, St. Louis, MO, USA) following manufacturer’s protocol, and the integrity was assessed by 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). RNA samples were used in array-based gene expression analysis only when the RNA Integrity Number was ≥7. Nucleic acid concentration and purity were assessed by NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).

Cells were washed in PBS then lysed using Tri-reagent (Sigma), and mRNA extracted by phase separation or RNeasy kit (Qiagen). Equal amounts of cDNA were synthesized using the High Capacity cDNA Kit (Applied Biosystems) and mRNA expression determined using Fast SYBR master mix in a StepOne thermocycler (Applied Biosystems) using the ΔΔCt method. The lung tissues were lysed in TRI Reagent (Sigma) using a gentleMACS dissociator and RNA extracted using the manufacturers protocol. cDNAs were synthesized using the Maxima H Minus cDNA Synthesis Kit (Thermo Scientific) and mRNA quantified using iTaq Universal SYBR Green Supermix (Biorad) and specific primers on a QuantStudio 3 RT-PCR system (Applied Biosystems).

The wild-type and plasmid-transformed C. necator H16 strains were grown in MM containing 4 mg/ml of fructose and 300 μg/ml of kanamycin. The cells were subcultured twice and grown aerobically for up to 24 h each time in 50-ml conical centrifuge tubes with orbital shaking at 30°C. Subsequently, the culture volume equivalent to an optical density at 600 nm (OD₆₀₀) of 1 for cells in exponential growth phase was pelleted by centrifugation at 13,000 × g for 1 min. The pellet was resuspended in 1 ml of TRI reagent (Sigma) and stored at −80°C. RNA was extracted using TRI reagent (Sigma) according to the manufacturer's recommendations. The samples were treated with 2 units of RQ1 RNase-free DNase (Promega), and 2 μg of total RNA was used for cDNA synthesis in a 20-μl reaction volume using the ProtoScript II first-strand cDNA synthesis kit (New England BioLabs). A real-time PCR analysis was performed in a 20-μl reaction volume using LuminoCt SYBR green qPCR ReadyMix (Sigma) and a Light Cycler 480 II (Roche). The relative quantification of mRNA levels was performed using the threshold cycle (ΔΔC_T) method (66 (link)). All biological samples were assayed in duplicates. Primers used for RT-PCR were P011_16S_f and P012_16S_r (for 16S RNA) and P013_yfp_f and P014_yfp_r (for the eyfp gene).

Individual zebrafish embryos (5 d.p.f.) from an in-cross of heterozygous carriers were homogenised in 100 μL of Tri Reagent (Sigma, Cat#93289) and transferred to 2 mL deep 96-well plates containing an additional 400 μL of Tri Reagent. The RNA-containing aqueous phase was stored at −80 °C until genotyping was completed. DNA was extracted from the interphase and organic phase according to the manufacturer’s instructions and genotyping was performed using the primers listed in Supplementary Table 4. Following genotyping, RNA was extracted from homozygous mutants and homozygous wildtype siblings. DNA was removed from RNA extraction using TURBO DNA-free kit (Invitrogen, Cat#AM1907). RNA was quantified using the Qubit RNA HS Assay Kit (ThermoFisherScientific, Cat#Q32852) and the Qubit 4 Fluorometer (ThermoFisherScientific, Q33226). RNA quality was checked by determining the 18S/28S rRNA ratio using the Fragment Analyzer RNA Kit (ThermoScientific, Cat#DNF-471-0500) and the 5200 Fragment Analyzer System (ThermoScientific, Cat#M5310AA). cDNA libraries were prepared from 1 μg of mRNA following poly-A selection using TruSeq stranded mRNA Library Prep (Illumina, Cat#20020595) according to manufacturer’s instructions.

To determine pri-miR expression and processing in cell culture, Huh7 cells were infected with HD Ads at a MOI of 100 infectious viral particles per cell. Two days after infection, cells were harvested and total RNA was extracted using Tri Reagent (Sigma, MI, USA). To determine pri-miR expression and processing in vivo, HBV transgenic mice [33 ] were used according to the protocols approved by the University of the Witwatersrand Animal Ethics Screening Committee. Mice were injected with a dose of 5 × 10⁹ HD Ad infectious viral particles via the tail vein. Animals were then killed at 1 week after injection and livers were harvested. Livers were homogenized and total RNA was isolated using Tri Reagent (Sigma, MI, USA). Thirty or sixty micrograms of RNA from cultured cells or liver tissue were analyzed using Northern blot hybridization according to previously described methods [11 , 13 (link)]. Labelled probes with sequences of 5′ CCG TGT GCA CTT CGC TTC 3′, 5′ CAA TGT CAA CGA CCG ACC 3′, and 5′ TAG GAG GCT GTA GGC ATA 3′ were used to detect mature guides 5, 8, and 9, respectively. Blots were then stripped and probed for U6 snRNA using a labelled oligonucleotide with sequence 5′ TAG TAT ATG TGC TGC CGA AGC GAG CA 3′. Band intensities were quantified by measuring photostimulated luminescence (PSL) using a FUJI FILM phosphorimager with Multi Gauge software.