Protocols: CD34+ bone marrow (Lonza) were sorted by CD34+CD38-GPRC5Cpos/neg into 384-well plates containing 240 nl of unique primer mix and 1.2 μl mineral oil (Sigma-Aldrich). Sorted plates were centrifuged at 2,200g for 5 minutes at 4°C and stored at −80 °C until further processing. Cells were lysed at 95C for 3 minutes. 160 nl reverse transcription reaction mix(NEB) and 2.2 μl second-strand reaction(NEB) mix was added per well to convert RNA into cDNA. cDNA from one plate was pooled together, and transferred to low DNA binding eppendorf tubes. 0.8x AMPure/RNAClean XP beads (Beckman Coulter) of total volume was used for cDNA cleanup. In vitro transcription was done with Transcription Kit (ThemoFischer) at 37C for 13h. To remove primers, with samples were incubated with EXO-SAP for at 94C for 3 minutes. RNA was fragmented by ncubating sample with fragmentation buffer (NEB) for 3 minutes at 94C. 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 11 PCR-cycles before Ampure bead (Beckman-Coultier) clean-up and subsequent quality control.
ENA first public
2022-01-01
ENA-LAST-UPDATE
2022-02-11
External Id
SAMEA7767647
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
2022-02-11T10:00:55Z
INSDC status
suppressed
Submitter Id
E-MTAB-9967:1289_P1_Gppos_002
broker name
ArrayExpress
cell type
hematopoietic stem cell (CD34+CD38-GPRC5Cpos)
common name
human
developmental stage
adult
disease
normal
individual
Patient 2
organism part
bone marrow
sample name
E-MTAB-9967:1289_P1_Gppos_002
sex
female