Vacufuge

Samples were collected from the solid deposits on a pre-filter of a commercially available domestic water purifier (referred to as pre-filter); and drying bed and sludge of the wastewater treatment plant (WWTP) of a dairy industry in Mumbai. The pre-filter was used for 3 months and the deposits are from about 2000 L of water. Dry samples (0.25 g) were processed using Power Soil DNA extraction kit (Mobio, USA) as per the manufacturer’s instructions. Fifteen-ml sludge sample was treated with 10% polyethylene glycol (PEG) 6000 overnight at 4 °C followed by centrifugation at 5000 g for 60 min and the virus-enriched precipitate was used for DNA extraction using the Power Soil DNA extraction kit (Mobio, USA). The total amount of DNA extracted was between 20 and 80 ng. DNA was further concentrated using Vacufuge (Eppendorf, Germany), and re-suspended in 10 µl DEPC treated water.

To dissolve proteins of the retinae, each isolated retina was incubated in 200 µL of 8 M aqueous urea solution for 60 min, and subsequently centrifuged for 5 min at 1400 g, as reported before [13 (link),34 (link)]. After collecting the supernatant, protein contents were estimated using a Microplate reader (BioTek Synergy H1 with Take3 plates, Agilent, Palo Alto, CA, USA), and the samples were aliquoted to contain 100 µg protein each. The volume of the aliquoted samples was adjusted to 100 µL with 25 mm NH₄HCO₃ solution followed by reduction with dithiothreitol (1 mM at 65 °C for 30 min) and carbamidomethylation (5 mM iodoacetamide at room temperature and in the dark for 30 min). After 9-fold dilution of the reduced and alkylated sample with aqueous 25 mM ammonium bicarbonate solution, sequencing-grade trypsin (2 µg) was added to digest the proteins overnight at 37 °C, after which the digestion was quenched with 5 µL of formic acid. The samples were desalted by solid-phase extraction using 1-mL Sep-Pak™ C-18 cartridges (Waters USA, Milford, MA), and then the extracts were dried under vacuum (Vacufuge™, Eppendorf AG, Hamburg, Germany) into 1.5-mL centrifuge tubes. The dried residues were dissolved in an aqueous medium containing 5% (v/v) and 0.1% (v/v) formic acid to produce 1 µg/µL protein content.

Brain cortices from 9-month-old WT, C3aR-KO, APP-KI, and APP-KI; C3aR-KO mice were snap frozen and ground using a bead beater. Ten milligrams of each sample was collected in 1.5 mL tubes and homogenized in 200 μL of 50 mM ammonium acetate solution. Samples were normalized based on protein concentrations from the BCA assay. Splash Lipidomix Mass Spec Standard (Avanti, 330707) was spiked (10 μL) in each sample before the extraction of lipids. Lipids were then extracted using methanol, methyl tert-butyl ether, and water. The extracted samples were dried in an Eppendorf Vacufuge and resuspended in 110 μL isopropanol/methanol (50:50, vol/vol). The samples were analyzed using a Vanquish UPLC and Lumos orbitrap mass spectrometer (Thermo Fisher Scientific). High-throughput analysis of lipidomic data was performed using Lipidsearch software (Thermo Fisher Scientific). The statistical analysis was done using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/MetaboAnalyst/ModuleView.xhtml).

The method of in vitro selection is similar to the one we reported previously (14 (link)). In brief, the initial library was obtained by ligating Lib-FAM-N₃₅ and Lib-rA. For each subsequent round, the library was produced by PCR. For each cleavage step, the DNA library was incubated with fresh Ln³⁺ solutions. For all the selections, the metal incubation time was maintained at 60 min. For the first four rounds, 50 μM Ln³⁺ was used while for the last three rounds, 10 μM Ln³⁺ was used. After incubation, the solution was mixed with 8 M urea and purified by 10% dPAGE (denaturing polyacrylamide gel electrophoresis). The position corresponding to the cleaved product was excised from the gel, the DNA was extracted by crushing and soaking the gel and was further desalted with a Sep-Pak C18 column (Waters). After drying in an Eppendorf Vacufuge at 30°C overnight, the dried DNA was re-suspended in 70 μl of 5 mM HEPES buffer (pH 7.5). The round 6 libraries for all the three selections were cloned and sequenced.