title: MPP
organism: Homo sapiens
description: Protocols: Blood was sampled through venous puncture. Sample collection was performed at 8.00-10.00 to avoid interference of circadian rhythms of immune parameters, and sample processing occurred within 2 hours. For sorting, BM-MNCs stored in liquid nitrogen were gently thawed in foetal calf serum (in house) containing 1.25mM MgCl2 and 0.1 mg/mL DNase I (Sigma). HSPCs were sorted by BD FACSAria II SORP flow cytometer sorter (Becton Dickinson HSPCs were defined as CD45+CD34+CD38dim cells, after exclusion of dead cells and doublets. Next, the lymphoid lineage was excluded in CD19-CD117+ cells. In CD45RAdimCD38+ cells, CMP, GMP, MEP and R1-3 progenitor populations were identified using CD123 and CD45RA expression. CD90 expression in CD38-CD45RA- cells determined MPP and HSC populations. HSCs, MPPs and GMPs populations were directly sorted in 100µL TRIzol (ThermoFisher) before processing for RNA sequencing. Total RNA was extracted using the standard TRIzol (ThermoFisher) protocol and used for library preparation and sequencing. CEL-Seq2-style primers were used (Hashimshony et al. 2016) to prime RT reaction on mRNA from cells, containing an Illumina 5' adapter as used in the illumina TruSeq small RNA kit.The Illumina 3' adapter from the small RNA kit was reverse transcribed during library prep using a random hexameter primer as described in the CEL-seq2 paper, after which the Illumina small RNA kit RT primer was used for cDNA synthesis from the amplified RNA.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-08-13T04:04:51Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2020-08-03T18:22:57Z
attributes.tag: External Id
attributes.value: SAMEA7116720
attributes.tag: INSDC center name
attributes.value: Twincore
attributes.tag: INSDC first public
attributes.value: 2020-08-13T04:04:51Z
attributes.tag: INSDC last update
attributes.value: 2020-08-03T18:22:57Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9399:MPP
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: hematopoietic multipotent progenitor cell
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: mixed
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: sample name
attributes.value: E-MTAB-9399:MPP
attributes.tag: scientific_name
attributes.value: Homo sapiens