Pooled sample scRN-seq: 10 types of immune cells
Peripheral blood mononuclear cells (PBMCs) were isolated from the phase above the red blood cells by density gradient centrifugation using LymphoprepTM (STEMCELL Technologies) as per the manufacturers instructions. The cells were counted via trypan blue stain and haemocytometer. Then, PBMC were suspended to 5*10^7 cells/mL. Ten types of immune cells were enriched and purified by EasySep Human Series kits based on MicroBeads positive and negative screening. The estimated content and experimental parameters of cells were shown in Table S1. the purification kit, surface marker of cells and theoretical purity were shown in Table S2. The purification process was conducted strictly according to the manufacturers instruction. Each enriched and purified cells were mixed with human universal antibody conjugated with a specific oligonucleotide sequence, named as Sample Tag for sample identification.GTTGTCAAGATGCTACCGTTCAGAG | Sample Tag sequence | AA...25...AA. The relationship between cell type and Sample Tag sequence was showed in Table S4. After mixture, 10 types of labeled cells constituted a mixed sample for single cell sequencing. Single cells were captured by micro-well and lysed, and mRNA was hybridized with primer sequences on Cell Capture Beads. The BD RhapsodyTM Targeted mRNA and Abseq Amplification Kit was used for library preparation. The libraries were sequenced on Illuminas sequencer.