Gene Expression
Nebulas
Gene Expression NebulasSummary: 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.
Overall Design: Transcriptional heterogeneity of LMPP, MLP and GMP human lymphomyeloid progenitors at the single cell level.
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| Growth Protocol: | 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}. |
| Treatment Protocol: | 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 |
| Extract Protocol: | 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 |
| Library Construction Protocol: | 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. |
| Molecule Type: | poly(A)+ RNA |
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| Library Layout: | PAIRED |
| Library Strand: | - |
| Platform: | ILLUMINA |
| Instrument Model: | Illumina HiSeq 4000 |
| Strand-Specific: | Unspecific |