GSM8003786: WT, snRNAseq; Mus musculus; RNA-Seq
To isolate single nuclei from mouse colon, Sigma's Nuclei EZ Prep Kit (Sigma, Cat #NUC-101) was used. Briefly, snap frozen mouse colon samples were firstly cut into 2mm pieces and homogenized using gentleMACS Dissociator (m_intestine_01 mode 2x, C Tubes) in 4 ml of ice-cold EZ Prep buffer (supplemented with 0.2 U/µl RNAse inhibitor, Clontech #2313A) and incubated on ice for 5 min. subsequently, nuclei were filtered through a 20-μm Pre-Separation Filter (Miltenyi Biotech, 130-101-812) and centrifuged at 500 × g for 5 min at 4 °C. After centrifugation, the nuclei were washed in 4 ml suspension buffer (1× PBS supplemented with 0.01% BSA and 0.2 U/µl RNase inhibitor (Clontech, Cat #2313A)). Isolated nuclei were resuspended in 1 ml suspension buffer, filtered through a 20-μm Pre-Separation Filter (Miltenyi Biotech, 130-101-812) and counted. A final concentration of 1000 nuclei per µl was used for 10x Genomics Chromium loading. Considering a 10–20% loss, we pipetted 9.9 μl single nuclei suspension (concentration of ~1000 nuclei/μl, ~9,900 nuclei), targeting the recovery of ~6,000 nuclei. A single nuclei suspension targeting 6000 cells per sample was loaded into 10x Genomics Chromium Single Cell Controller and barcoded with unique molecular identifiers. Single-nuclei RNA-seq libraries were obtained following the 10x Genomics recommended protocol, using the reagents included in the Chromium Single Cell 3′v3.1 Reagent Kit Libraries.