title: Population RNA Seq of long-term HSCs isolated from mice after myocardial infarction (day 2 post infarct) or sham surgery and at-RA treatment or DMSO control treatment
design: Population RNA Seq of long-term HSCs isolated from mice after myocardial infarction (day 2 post infarct) or sham surgery and at-RA treatment or DMSO control treatment
library_construction_protocol: Myocardial infarction was induced in female C57BL/6J mice aged 6 to approximately 12 weeks through permanent occlusion of the left anterior descending artery (LAD). Mice were intraperitoneally injected on the 1st and 2nd day after MI surgery with either 30 mg/kg body weight at-RA (Sigma-Aldrich; MI+at-RA condition), or with the corresponding amount of DMSO in phosphate-buffered saline (PBS) (MI+vehicle condition). The mice were euthanized for analysis two days after MI. Murine bone marrow (BM) cells were isolated from the femur, tibia, hip bone and vertebrae by gentle crushing with mortar and pestle in PBS. Red blood cell lysis was performed with ACK Lysing Buffer (Thermo Fisher Scientific) for 5_min at room temperature (RT). Dynabeads Untouched Mouse CD4 Cells Kit (Invitrogen) was used for lineage negative enrichment according to the manufacturerÕs protocol. Briefly, the BM was stained with 1:4 dilution of the Lineage Cocktail for 30Ð60_min at 4_¡C on a rotating wheel. Labelled cells were then incubated for 20_min with 400_µl of washed Dynabeads coated with polyclonal sheep anti-rat IgG per sample. Depletion of lineage cells was performed using a magnet. Lineage-depleted BM cells were stained with lineage markers (Gr1, CD11b, B220, Ter119, CD4 and CD8a), ckit, Sca1, CD150, CD48 and CD34. Sorting was then performed using a FACS Aria II, III or FACSymphony (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany. Each 1500 LT-HSCs (Lin-negative, Sca1-positive, c-Kit-positive, CD150-positive, CD48-negative, CD34-negative) of sham+vehicle, MI+vehicle or MI+ at-RA mice were sorted, respectively, in 3 biological replicates. Cells were sorted into RNA lysis buffer (Arcturus PicoPure RNA Isolation Kit (Applied Biosystems)) and then stored at -80¡C until further use. RNA isolation from LT-HSCs 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 LT-HSCs,12 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.