title: Population RNA-seq of young hematopoietic stem cells (HSCs) vs young progenitors vs old HSCs isolated from human bone marrow
design: Population RNA-seq of young hematopoietic stem cells (HSCs) vs young progenitors vs old HSCs isolated from human bone marrow
library_construction_protocol: Human frozen bone marrow samples were thawed at 37oC and transferred to 10ml of thawing media (IMDM, 10% FCS, 1mM EDTA, 1:1000 DNase) and spun down for 8 min at 300g. Cells were resuspended in Stem Span and let for recovery in the incubator for 20 min. Cells were resuspended in 500ul antibody mix (Lin(CD3/14/16/19/20/56)-APC; CD38-Pe/Cy7, CD34-FITC*)  at 4oC for 45 min. Cells were washed with 0.9% NaCl and incubated with Zombie-Aqua (1:1000) for 10 min at RT. Cells were finally washed and resuspended in 0.9% NaCl and filtered for FACS sorting. Sorting was then performed using a FACS Aria III at the MPI FACS facility, Freiburg, Germany.  1000 cells of young HSCs/Progenitors and old HSCs were sorted in 4 and 7 biological replicates, respectively. Cells were sorted into RNA lysis buffer (Arcturus PicoPure RNA Isolation Kit (Applied Biosystems)) and then stored at -80οC until further use. RNA isolation was conducted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) following the manufacturer's guidelines. DNAse treatment was carried out employing the RNase-Free DNase Set (Qiagen). The resulting total RNA was utilized for generating cDNA libraries. cDNA libraries were generated using SMARTseq v4 (Takara Bio). For HSCs, 13 cycles of amplification were performed. To produce uniquely- and dually-barcoded sequencing libraries from the cDNA libraries, the NEBNext Ultra II FS DNA library kit (NEB) was utilized. This involved fragmenting 5 ng of the cDNA library for 22.5 minutes, followed by adaptor ligation and library amplification using cycle numbers determined by amount of input material.