Protocols: CD34+ cells were isolated using EasySep Human Cord Blood CD34 Positive Selection Kit II (StemCell Technologies) from mobilized peripheral blood. Isolated cells were immediately stained with CD34/CD38, and sorted for the CD34+CD38- HSCs on the FACS Aria III (Becton Dickinson). After 24 hours of prestimulation in culture, the cells were transduced with lentivirus of GPR-OE and control. 2 days post-transduction, positively transduced cells which were marked by GFP are sorted using the FACS Aria III (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany. The following sort parameters were used: 100 um nozzle with approximately10,000 evt/s. 3000 GFP+ cells were collected into ice-cold RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen)) and stored at -80C until further usage. Total RNA isolation was using ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen) according to the manufacturer's instructions. DNAse treatment was performed using RNAse-free DNAse Set (Qiagen). Total RNA was used as starting material for cDNA-libraries preparation using the SMARTseq v4 Ultra Low RNA Kit (Takara) according to the manufacturers indications. Sequencing libraries were prepared from 10ng of cDNA libraries as input using NEBNext Ultra II FS DNA library prep kit (#E7805S, NEB) according to manufacturer's protocol together with unique dual indexing barcodes (NEB, #E6440). Libraries were amplified with 7 PCR-cycles before Ampure bead (Beckman-Coultier) clean-up and subsequent quality control.
ENA first public
2022-01-01
ENA last update
2022-01-01
External Id
SAMEA7699362
INSDC center alias
Max Planck Institute of Immunobiology and Epigenetics (MPI-IE)
INSDC center name
Max Planck Institute of Immunobiology and Epigenetics (MPI-IE)
INSDC first public
2022-01-01T00:12:38Z
INSDC last update
2022-01-01T00:12:38Z
INSDC status
public
Submitter Id
E-MTAB-9874:Empty_2
broker name
ArrayExpress
cell type
CD34-positive, CD38-negative multipotent progenitor cell
common name
human
developmental stage
adult
disease
normal
genotype
wild type genotype
individual
1
organism part
blood
sample name
E-MTAB-9874:Empty_2
sex
male
specimen with known storage state
frozen specimen (-80 degree celsius)
stimulus
mobilization