概要: We performed single cell RNA sequencing of human haemopoietic lympho-myeloid progenitors with the aim to separate functionally and transcriptionally distinct progenitors within the heterogeneous GMP, LMPP and MLP populations. We performed RNA sequencing in a total of 415 single cells. From donors 1 (CB369) and 2 (CB431), 163/166 and 157/249 cells passed quality control.We further analysed the 320 single cells that passed quality control from two different donors (157 and 163 from each donor; 91 LMPP, 110 MLP and 119 GMP). We performed clustering using Seurat package, which identified 3 clusters of cells. We identified genes that were differentially expressed among cells of the different clusters. The majority of the cells in cluster 1 were MLP, the majority of cluster 3 were GMP while cluster comprised of LMPP and GMP cells. Cluster 1 showed high expression of lymphoid-affiliated genes. Conversely, cluster 3 showed increased expression of myeloid genes, while cluster 2 showed a mixed transcriptional signature. PCA analysis revealed a transcriptional continuum of LMPP, MLP and GMP populations.
项目整体设计: Transcriptional heterogeneity of LMPP, MLP and GMP human lymphomyeloid progenitors at the single cell level.
Fresh cord blood from normal donors were obtained with informed consent. They were processed within 16-34h after collection. Mononuclear cells were isolated and CD34+ fraction was separated as described{Quek et al, JouRNAl of Experimental Medicine 2016}.
处理方案:
Fresh Cord Blood cells were obtained and processed with ficoll to obtain mononuclear cells. CD34+ cells were purified using CD34 Microbead Kit and MACS separation columns
提取方案:
Single cells were deposited in individual wells of 96 well plates, directly lysed according to the SmartSeq2 protocol and stored at -80C. No extraction protocol was performed
建库方案:
Single cell libraries for RNAseq were prepared using SmartSeq2 protocol (Picelli et al., -ture Protocols 2014) and the Illumina Nextera XT Index Kit (96 indexes, 384 samples, FC-131-1002) for cDNA tagmentation and indexing. ERCC RNA Spike-In Mix (Ambion, 4456740) are included.
测序信息
分子类型:
poly(A)+ RNA
库的片段类型:
PAIRED
库的链类型:
-
测序平台:
ILLUMINA
测序仪型号:
Illumina HiSeq 4000
链特异性:
Unspecific
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文章
Single-cell RNA-seq highlights intratumoral heterogeneity in primary glioblastoma.
Science (New York, N.Y.) . 2014-06-12 [PMID:
24925914]
