GSM8072127: WT Spleen CD45+ scRNAseq; Mus musculus; RNA-Seq
Splenocytes were collected through mashing the spleen by using the plunger end of the syringe in 1 ml Facs buffer through placing the spleen into the cell strainer. Bone marrow cells were flushed from the femur and tibia bones. Peripheral blood (600–800µl) was collected by retro-orbital bleeding into tubes (BD 365965). Cells were then filtered through a 40μm cell strainer. Cells were centrifuged for 5 min at 500g. The red blood cells were then lysed with RBC lysis buffer for 3 min at room temperature. After centrifugation, cells were washed twice with Facs buffer and resuspended in Facs buffer. Mouse colons were surgically excised, flushed with PBS, then opened longitudinally and cut into approximately 0.5-cm-long pieces. The colon tissue was washed again with 10 ml PBS followed by incubation in 20 ml HBSS buffer containing 2% FBS, 1mM EDTA and 1mM DTT in 50 ml canonical tube, at 37 °C with constant shaking at 180 rpm for 20 min to release epithelial cells. The colon tissues were then washed with 10 ml HBSS buffer before being digested in 10 ml 1640 buffer containing 2% FBS, collagenase type II (2mg/ml), collagenase D (0.5mg/ml), DispaseII (3mg/ml) and DNase I (0.1mg/ml) at 37 °C with constant shaking at 180 rpm for 30 min in a 15ml tube. After digestion, the digested cells and remaining tissue fragments were vortexed intensely for 20 s before being passed through a 40 μm cell strainer. The filtered cell suspension was centrifuged at 600g, 4 °C for 5 min, then resuspended in 1 ml Facs buffer (2% FBS, 2mM EDTA in DPBS). Viable cells were used for sequencing following the 10× Genomics protocol. Libraries were prepared using the Single Cell 5′ Library Kit V2 (10× Genomics). Transcriptome profiles of individual cells were determined by 10× Genomics-based droplet sequencing and libraries were sequenced with paired-end reads on an Illumina NovaSeq 6000