GSM8684489: Slc12a1_INTACT_Medulla; Mus musculus; RNA-Seq
Kidney was dissected out after PBS washout via aortic perfusion and snap-frozen in liquid nitrogen immediately after dissection. Frozen tissue was stored at -80 °C until subsequent tissue processing. Nuclei were isolated using the 10X Chromium Nuclei extraction kit according to the manufacture protocol. The resulting pellet was resuspended in 10 ml NSB (10mL DPBS + 10µl RNasin Plus). The nuclei suspension was mixed with 5µl Vybrant Ruby stain and incubated on ice for 15 min. The nuclei were sorted in 500 µl final resuspension buffer (FSB, 1ml DPBS with 1% BSA + 5 µl Protector RNase inhibitor) with a low flow rate and pressure to ensure high viability. Two main gates were used. A 561+683 emission for the ruby stain and a 488 530/40 emission for GFP. Low trigger pulse width was used as singlet discriminator. One hundred and fifty thousand nuclei were collected and centrifuged at 500g for 10 min at 4°C in a swinging bucket rotor. The top supernatant was carefully removed, and 6 µl volume was left to resuspend the nuclei pellet. Single nucleus RNA-seq was performed using a PIPseq™ T10 3ʹ Single Cell RNA Kit v5.0 (Fluent BioSciences). Single nucleus suspensions with a maximal concentration of 3,400 nuclei/uL (for a total of 17,000 nuclei per sample) were added directly into Particle-templated Instant Partition (PIP) tubes. Following the manufacturers protocol, with subsequent vortexing the nuclei were partitioned into PIPs. Following capture and lysis of the RNA and downstream cDNA generation and amplification, the resulting cDNA was run out 2% gel electrophoresis for approximate band size (greater than 500bp). The cDNA was then used as a template for library preparation according to the reagent kit protocol. https://www.fluentbio.com/resource-category/user-guides/. The libraries were profiled on a Bioanalyzer NanoChip (Agilent), and then subsequent sequencing was performed on a NextSeq2000 (Illumina).