GSM7882765: MI-NS, scRNAseq; Mus musculus; RNA-Seq
The samples were fully lysed using a lysis buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl2, and 0.05% (v/v) NP-40 detergent) and were then filtered by a 30 μm cell strainer. After pelleting, the nuclei were reconstituted in a wash buffer (10 mM Tris [pH 7.4], 10 mM NaCl, 3 mM MgCl2, 1% BSA, 1 mM DTT, RNase inhibitor 1U/µl, nuclease-free water) and combined with 25% optiprep and layered on a 29% optiprep cushion. After centrifugation, the nuclei were reconstituted with the wash buffer again. Finally, the nuclei were resuspended into the nuclei resuspension buffer (nuclei buffer (10XGenomics, 20x) 1x, 1 mM DTT, RNase inhibitor 1U/µl, nuclease-free water) with a concentration of ~1x10^6/ml, and the single-cell nuclei suspension was prepared. Once the extracted nuclei passed the quality test, single-cell RNA sequencing (RNA-seq) libraries were prepared with Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 on the Chromium Controller (10 × Genomics). Single-nuclei gel beads in the emulsion (GEMs) were produced as directed by the manufacturer. The released RNA of single nuclei was barcoded in each GEM via reverse transcription. Sequencing was performed with a depth of at least half a million reads/cell and 150 bp paired-end reads (PE150) using the Illumina NovaSeq 6000.