GSM9856158: Manf+/+; Mus musculus; RNA-Seq
For scRNA-seq analysis of CD45+ immune cells from mouse intestine, colonic tissues were harvested, washed with ice-cold PBS, and enzymatically digested with Collagenase II (Sigma, V900892), Dispase II (Roche, 04942078001), and DNase I. Cell number and viability were assessed using a SeekMate Tinitan Fluorescence Cell Counter (SeekGene, M002C) with AO/PI reagent. Depending on sample quality, debris and dead cells were removed using the Debris Removal Kit and Dead Cell Removal Kit (Miltenyi Biotec, 130-109-398 and 130-090-101), respectively. CD45+ cells were then enriched by magnetic labeling with CD45 MicroBeads (Miltenyi Biotec, 130-052-301). Isolated cells were washed twice with RPMI 1640 medium and resuspended at 1 × 106 cells/mL in RPMI 1640 supplemented with 2% FBS. ScRNA-seq libraries for mouse CD45+ immune cells were prepared using the SeekOne® DD Single Cell 3′ Library Preparation Kit (SeekGene, K00202) according to the manufacturer's instructions. Briefly, cells were mixed with reverse transcription reagents and loaded into the sample well of the SeekOne® Chip S3, followed by the addition of barcoded hydrogel beads and partitioning oil into the corresponding wells. After droplet generation, reverse transcription was performed at 42°C for 90 min and inactivated at 85°C for 5 min. cDNA was then recovered, purified, amplified, fragmented, end-repaired, A-tailed, and ligated to sequencing adapters. Indexed libraries were purified using VAHTS DNA Clean Beads (Vazyme, N411-01), analyzed using Qubit (Thermo Fisher Scientific, Q33226) and a Bio-Fragment Analyzer (Bioptic, Qsep400), and sequenced on the GeneMind SURFSeq 5000 platform with paired-end 150 bp reads.