GSM8031211: f_term,replicate 1,scRNAseq; Homo sapiens; RNA-Seq
Peripheral blood samples were collected from 3 full-term, 3 preterm and 3 adult controls. Single-cell suspensions of peripheral blood mononuclear cells (PBMCs) were obtained by centrifuging human blood samples on a Ficoll gradient. Briefly, blood was mixed with an equal volume of 2% FBS in PBS and gently layered on the Ficoll gradient. Cells were centrifuged at 1000 x g for 25 min, without braking. The cells in the middle layer were then washed once with PBS and red cells were removed using ACK lysing buffer, resuspended in 2% FBS in PBS for use. The PBMC suspensions were stained with surface markers at 30 min at 4°C in dark. The strategy for MDSCs was CD11b+HLA-DR-/lo. Live immature myeloid cell in RPMI1640 supplemented with 20% FBS were sorted in a BD FACSAria III cell sorter (BD Biosciences). Droplet-based scRNA-seq library praperarion: single cells were captured via the GemCode Single Cell Platform using the GemCode Gel Bead, Chip and 30-end Library Kits (103 Genomics), according to the manufacturer's protocol. Flow sorted CD11b+HLA-DR- cell which were pre-labeled with hashtag antibodies (Biolegend), were pooled and loaded at 60,000 cells per channel. The cells were then partitioned into the GemCode instrument, where individual cells were lysed and mixed with beads carrying unique barcodes in individual oil droplets. The products were subjected to reverse transcription, emulsion breaking, cDNA amplification, and sample index attachment