Protocols: 8-10 week old Gprc5c-KO mice and corresponding controls were sacrificed. Murine bone marrow (BM) cells were isolated from the femur, tibia, hip bone, and vertebrae. Lineage positive cells were depleted from the bone marrow, and the removing cells were stained for lineage markers, cKit, Sca1, CD150, CD48 and CD34. The Lineage-cKit+Sca1+CD150+CD48-CD34- (HSCs) were sorted on the FACS Aria III (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany. The following sort parameters were used: 70 m nozzle; 10,000 evt/s.; 70 psi. 2000 sorted HSCs were collected into chilled RNA lysis buffer (ARCTURUS PicoPure RNA Isolation Kit (Life Technologies, Invitrogen)) and stored at -80C until further usage. Total RNA isolation was performed 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
SAMEA7699190
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-9862:HSC_Ctrl_2
age
8
broker name
ArrayExpress
common name
house mouse
developmental stage
adult
genotype
wild type genotype
individual
Mouse 2
organism part
bone marrow
sample name
E-MTAB-9862:HSC_Ctrl_2
sex
male
specimen with known storage state
fresh specimen
strain
C57BL/6J