Population RNA Seq of enriched human peripheral blood monocytes treated with at-RA/4-oxo-RA/DMSO for 24h in culture
Population RNA Seq of enriched human peripheral blood monocytes treated with at-RA/4-oxo-RA/DMSO for 24h in culture
Human peripheral blood mononuclear cells enriched for monocytes isolated from sternal bone marrow of myocardial infarction patients or control patients were sorted into low-attachment 96-well plates containing StemSpan SFEM II media (StemCell Technologies) supplemented with StemSpanª CD34+ Expansion Supplement (StemCell Technologies). For in vitro cultivation, at-RA (2.5 M final concentration), 4-oxo-RA (2.5 M final concentration), or the respective volume of DMSO was added to the culture medium. Cells were cultured for 24h prior to harvest for RNA extraction. Mononuclear cells were isolated from the peripheral blood of healthy individuals and enriched for monocytes using the EasySep Human Monocyte Enrichment Kit (StemCell Technologies). After culturing the cells, 96-well plate was centrifuged at 400g for 5 minutes. The cell culture medium was carefully removed using a pipette, and the cells were subsequently extracted into lysis buffer (Arcturus PicoPure RNA Isolation Kit (Applied Biosystems)). Cells were then stored at -80¡C 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). 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.