Single cell RNA-seq of cattle: adult female Duodenum (replicate2)
The cell/nucleus suspension (300-600 living cells per microliter determined by Countstar) was loaded onto the Chromium single cell controller (10 Genomics) by Single Cell 3 Library and Gel Bead Kit V3.1 (10 Genomics) and Chromium Single Cell G Chip Kit (10 Genomics) to generate single-cell/nucleus gel beads in the emulsion. In short, single cells/nuclei were suspended in PBS with 0.04% BSA. About 4,000 cells/nuclei were added to each channel, and the target cells/nuclei recovered were estimated to be about 2,000 cells/nuclei. Captured cells/nuclei were lysed and the released RNA was barcoded through reverse transcription in individual Gel Bead-In Emulsions (GEMs). Reverse transcription was performed on an S1000TM Touch Thermal Cycler (Bio-Rad) at 53C for 45 min, followed by 85C for 5 min, and held at 4C. The cDNA was generated, amplified, and quality assessed using an Agilent 4200. The libraries were finally sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with a pair-end 150 bp (PE171) strategy (CapitalBio Technology Co., Ltd., Beijing, China)