Purelink kit

Bacterial DNA was extracted as described previously (Meisel et al., 2016 (link)) using the Invitrogen PureLink kit. PCR and sequencing of the V1V3 hypervariable region was performed using 300-bp paired end chemistry and barcoded primers (27F, 534R) on the Illumina MiSeq platform. Accuprime High Fidelity Taq polymerase was used for PCR cycling conditions: 94 °C for 3 min; 35 cycles of 94 °C for 45 sec, 50 °C for 60 sec, 72 °C for 90 sec; 72 °C for 10 min. PCR products were purified using the SequalPrep kit (Invitrogen), according to manufacturer’s instructions, and pooled in equal amounts for sequencing. For bacterial load comparisons, 16S rRNA genes were amplified by qPCR using Fast SYBR Green Master Mix and the optimized primers 533F, 902R. Samples were compared to standard curves generated from known concentrations of serially diluted bacterial DNA to calculate burden.

For donors 721171, 311552, and 271865, total cellular RNA was isolated using a Purelink kit (Invitrogen, Carlsbad, CA). Genomic DNA was eliminated with TURBO DNA-free Kit (Invitrogen). Samples were analyzed using a bioanalyzer and sequencing libraries prepared with SMART-Seq v4 Ultra Low Input Kit for Sequencing (Takara Bio USA, Mountain View, CA).
For donors 201920 and 205007, cell pellets were flash frozen and subsequently total cellular RNA was isolated using an RNeasy kit (Qiagen, Germantown, MD). Purity was assessed using a bioanalyzer and sequencing libraries prepared with SMARTer Stranded Total RNA-Seq Kit V2 Pico Input Kit (Takara).

The full-length hCB1R gene (1.4 kbp) was amplified from pRC/CMV CB1 construct as template, using Pfu DNA polymerase (Stratagene) under the following thermocycling conditions: 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s and 68 °C for 2 min, followed by an extension time of 5 min at 68 °C in an Eppendorf Mastercycler (Westbury, NY). NheI and BamHI restriction sites were incorporated into forward 5′-CGCTAGCATG-AAGTCGATCCTAGATGGCCT-3′ and reverse 5′-TATGGATCC-TCACAGAGCCTCGGCAGACGTG-3′ primers, respectively. The PCR product was purified using a MinElute PCR kit (Qiagen) and was digested using BamHI and NheI restriction enzymes. The same restriction enzymes were used for digestion of pcDNA5/FRT expression vector (Invitrogen). The vector and PCR fragment were purified using a MinElute kit and ligated at room temperature for 1 h. The ligated products were then transformed into One Shot Top10 competent E. coli cells following the vendor’s protocol (Invitrogen). Plasmid preparations were cultured in Luria broth containing 0.1 mg/mL ampicillin. Recombinant plasmid DNA was isolated using a pure link kit (Invitrogen), and DNA insertion was confirmed by sequencing (University of Connecticut Biotechnology Center, Storrs, CT).

RNA was isolated from day-25 kidney organoids using the PureLink Kit (Invitrogen). RNA was then reverse transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real-time quantitative PCR (RT-qPCR) reactions for APOL1 (Hs01066280_m1; Thermo Fisher) and ACTB (Hs01060665_g1) were run in duplicate on the QuantStudio 5 Real-Time PCR System (Applied Biosystems). Relative APOL1 expression was calculated using the 2^−ΔΔCT method. Statistical significance was tested using two-way ANOVA in Graphpad (Prism).

RAW264.7 cells were incubated in cell culture media with normal [Na⁺] or low [Na⁺] with cytokines for 24h with at least 3 biological replicates in each group. Total RNA was extracted using PureLink kit (Invitrogen, Carlsbad, CA) according to manufacturer’s instructions, and purified using the Rneasy Mini Kit (Qiagen, Germantown, MD). Agarose gel electrophoresis was used to check the integrity of total RNA.