GSM8722606: Fish 4 Kidney, 1.5 month juvenile fish; Danio rerio; RNA-Seq
Nuclei were isolated with Nuclei EZ Lysis buffer (NUC-101, Sigma) supplemented with protease inhibitor (5892791001, Sigma) and RNase inhibitors (N2615, Promega; AM2696, Life Technologies). Zebrafish brain, muscle and kidney tissue were dissected and quickly rinsed with ice-cold PBS before snap-froze. Samples were minced into <2-mm pieces with 500 µL of ice-cold NLB buffer (10 mL Nuclei EZ lysis buffer supplemented with 1 tablet of protease inhibitor and 10 µl of each RNase inhibitors) and incubated on ice for 5 min with an additional 500 µl of NLB. The tissue and NLB buffer mix was then transferred to the Dounce tissue grinder (D8938, Sigma), gently grind 30 times with Pestle A and 15 times with Pestle B. The homogenate was then incubated on ice for an additional 5 min. The homogenate was filtered through a mini-40-µm and a subsequent mini 20-µm cell strainer (43-10040-50 & 43-10020-50; PluriSelect) and then centrifuged at 500 x g for 5 min at 4°C. The pellet was resuspended and washed twice with 1 mL of NLB buffer to remove residue cytoplasmic RNAs. After another centrifugation, the nuclei pellet was resuspended in Nuclei Suspension Buffer (1XPBS, supplemented with 2% BSA and 2 µl Promega RNase inhibitor), filtered through a mini-10-µm cell strainer (43-10010-50, PluriSelect). Nuclei were counted on hemocytometers (KIMA211710, Vetriplast-10). The quantified brain and muscle nuclei were pooled together in 1:2 ratio, kidney nuclei were processed on its own. The nuclei were partitioned into each droplet with a barcoded gel bead using the 10X Chromium instrument with the Chromium Single Cell 3′ Reagent Kit v.3.1 to generate transcriptome libraries.