GSM5289682: pool1; Mus musculus; RNA-Seq
Tissue was crushed on liquid nitrogen and a small amount was transferred to a non-stick Eppendorph tube and was added 1.5 mL lysis buffer (Pure lysis buffer (L9286-180ml, Sigma) w. 0.1% DTT (43816-10ml, Sigma) and 1% Triton (T1565-1.7ml, Sigma)). Samples were incubated on ice for 5 min and were carefully pipetted up and down during the incubation. Samples were centrifuged for 5 min at 750 g at 4 °C and the supernatant was removed. Pellet was resuspended in 1 mL nuclei buffer (PBS, w/o MgCl₂ and CaCl₂ (20012068, Invitrogen), 1% BSA (SRE0036-25ml, Sigma), 2 mM MgCl2 (M1028-100ml, Sigma) and 0.1 % Protector RNase inhibitor 40U/µl (3335399001, Sigma), and solution was passed through a 40 µm strainer (43-10040-40, Pluriselect). Samples were incubated for 10 min on ice and centrifuged for 5 min at 750 g at 4 °C. Supernatant was removed and the pellet was resuspended in 110 µL nuclei buffer. For each sample, the remaining 100 µL were added 1 µL of individual TotalSeq™-A antibodies (BioLegend, San Diego, CA) from 0.5 µg/µl stocks to enable multiplexing . Samples were incubated for 30 min on ice, and each sample was added 1 mL nuclei buffer. Samples were centrifuged 10 min at 1000 g at 4 °C, and the supernatants were discarded. Pellets were resuspended in 200 µL Nuclei Freeze Buffer (EZ Nuclei Storage Buffer (NUC101, Sigma), 0.1% BSA (SRE0036-25ml, Sigma) and 2 mM MgCl2 (M1028-100ml, Sigma)) and were stored at -80 °C until sorting. Using a SH800S Cell Sorter (SONY) 4,250 nuclei from each of the 6 samples was sorted and pooled for generation of libraries for snRNA-seq using the 10x Genomics Chromium single-cell 3′ reagent kits (version 3.1).