title: GSM7074904: ORG-PT_cut-and-run_IgG (HNF4A control); Homo sapiens; OTHER
library_construction_protocol: Frozen human adult kidney cortex was cut into < 1 mm pieces, homogenized using a dounce tissue homogenizer, and incubated on ice for 3 min in chilled Nuclei EZ lysis buffer supplemented with protease inhibitor cocktail. The homogenate was filtered through a 40-μm cell strainer and centrifuged at 500 g for 5 minutes at 4°C. The pellet was resuspended and washed with chilled lysis buffer, and further incubated on ice for 3 minutes. Following centrifugation, the pellet was resuspended in XL wash buffer, and filtered through a 5-μm cell strainer. 500,000 isolated nuclei were fixed with 0.5% formalin for one minute at room temperature, followed by addition of glycine.  Sorted kidney organoid-derived proximal tubular cells were fixed with 0.5% formalin for one minute at room temperature. After inactivating unreacted aldehyde by adding glycine, 100,000 cells were treated with chilled multiome lysis buffer on ice for 5 minutes to obtain nuclei. Libraries were generated with the CUTANA™ ChIC/CUT&RUN Kit (EpiCypher, 14-1048). Fixed nuclei were incubated with Concanavalin A (ConA) conjugated paramagnetic beads and stained with 0.5 µg of antibodies. The remaining steps were performed according to manufacturer's instructions. Library preparation was performed using the NEBNext Multiplex Oligos for Illumina with manufacturer's instructions, including minor modifications indicated by CUTANA described above. CUT&RUN libraries were sequenced on a NovaSeq instrument (Illumina, 150 bp paired-end reads).