Glycogen

Single His2Av-RFP(III), brat^Df/fs1; His2Av-RFP(III) or brat heterozygous; His2Av-RFP(III) embryos (3–4 replicates per time point) were dechorionated in 100% bleach for 1′, mounted in halocarbon (700) oil (Sigma Aldrich, St. Louis, MO) on a coverslip, and imaged using a Nikon Ti-2e Epiflourescent microscope using a 60× objective. The nuclear cycle was identified following mitosis based on His2Av-RFP marked nuclear density (calculated by the number of nuclei/2,500 μm²). At the indicated time, embryos were picked into Trizol (Invitrogen, Cat #15596026) with 200 μg/ml glycogen (Invitrogen, Cat #10814010) and pierced with a 27G needle to release the RNA for 5 min. Late-stage single oocytes (4 replicates per genotype) were dissected from the ovaries of the mothers of the respective genotype and staged based on morphology. The oocytes were picked into Trizol (Invitrogen, Cat #15596026) with 200 μg/ml glycogen (Invitrogen, Cat #10814010) and pierced with a 27G needle to release the RNA for 5 min. RNA was extracted and RNA-seq libraries were prepared using the TruSeq RNA sample prep kit v2 (Illumina, San Diego, CA). Seventy-five base pair reads were obtained using an Illumina NextSeq500 sequencer at Northwestern Sequencing Core (NUSeq Core). This protocol is expanded in McDaniel and Harrison (2019) (link).

Human umbilical vein endothelial cells were crosslinked with 50 mJ UV light and treated in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.5% NaCl, 0.5% NP-40, 80 U Ribolock protease inhibitor (Thermo Fisher) and volumes were adjusted to 1 ml. Streptavidin-coupled beads C1 (Thermo Fisher) were pre-blocked using ytRNA (Thermo Fisher, AM7119) and glycogen (Thermo Fisher, R0561). For selection of RNP complexes, lysates were pre-cleared with 50 μl pre-blocked beads for 2 h at 4°C and subsequently incubated with 100 pmol 2’O-MeRNA oligonucleotides targeting Aerrie or a scrambled control oligonucleotide for 1 h at 37°C. Sequence of the oligos are listed in the Supplementary Table 2. RNP-oligonucleotide complexes were captured using 25 μl pre-blocked (yeast tRNA, glycogen; both 0.2 mg/ml) streptavidin C1 beads (Thermo Fisher) for 1 h at 37°C. Beads were washed thoroughly with washing buffer (50 mM Tris–HCl pH8, 150 mM NaCl, 0.05% NP-40) and eluted with biotin (250 mM) for 1 h at RT. Eluates were analyzed by RT-qPCR and mass spectrometry.

Pure bacterial EV preparations were resuspended in TRIzol LS (Thermo Fisher Scientific), 200 μL of chloroform, and 2 μL of 5 mg/mL glycogen (Thermo Fisher Scientific) added. Samples were vortexed for 15 s, incubated for 10 min on ice, and centrifuged at 13,000 × g for 10 min at 4°C. The upper aqueous phase was collected and mixed with 1.25 volumes of 100% ethanol, and RNA purified using mirVana RNA isolation kit (Thermo Fisher Scientific), according to the manufacturer’s protocol for total RNA isolation. Cultured 5637 human bladder carcinoma cells in TRIzol (Thermo Fisher Scientific) were mixed with 50 μL of miRNA Homogenate Additive (mirVana RNA isolation kit), 100 μL of chloroform, and 2 μL of 5 mg/mL glycogen. Samples were vortexed and incubated for 10 min on ice to continue with protocol as described above for extractions from bacterial EVs. RNA yields were determined by a Qubit 2.0 fluorometer using Qubit RNA HS assay kit (Thermo Fisher Scientific).