title: Population RNA Seq of cardiac fibroblasts, cardiac endothelial cells, cardiac monocytes, cardiac macrophages - isolated from mice after at-RA treatment or 4-oxo-RA or DMSO control treatment (24h post injection)
design: Population RNA Seq of cardiac fibroblasts, cardiac endothelial cells, cardiac monocytes, cardiac macrophages - isolated from mice after at-RA treatment or 4-oxo-RA or DMSO control treatment (24h post injection)
library_construction_protocol: Female C57BL/6J mice aged 12 weeks were intraperitoneally injected with either 30 mg/kg body weight at-RA (Sigma-Aldrich) (at-RA condition), 30 mg/kg body weight 4-oxo-RA (Sigma-Aldrich) (4-oxo-RA condition), or with the corresponding amount of DMSO in PBS (vehicle condition). The mice were euthanized for analysis 24h after injection. Hearts were extracted and myocardial tissue was excised, minced and subjected to enzymatic digestion using a mixture of collagenase I (450 U/ml), collagenase XI (125 U/ml), DNase I (26 U/ml), and hyaluronidase (60 U/ml) (all from Sigma-Aldrich). Enzymatic reaction was incubated at a temperature of 37¡C and a rotation speed of 600 rpm for a duration of 1 hour and subsequently stopped by the addition of 30 ml of FACS buffer consisting of phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin and 1% fetal bovine serum. Cardiac cell suspension was stained using the following antibodies: Ly6C-FITC, CD115-BV711, B220-BV650, CD3-PerCP-Cy5.5, Ly6G-APC, CD11b-APC-Cy7, CD45.2-PB, and F4/80-Pe-Cy7. Sorting was then performed using a FACS Aria II, III or FACSymphony (Becton Dickinson) at the MPI FACS facility, Freiburg, Germany.  1000 monocytes (CD45 positive, CD11b positive, F4/80 negative, LyC6 positive), 5000 endothelial cells (CD45 negative, CD31 positive), 5000 macrophages (CD45 positive, CD11b positive, F4/80 positive, LyC6 negative) or 5000 fibroblasts (CD45 negative, GP38 positive) of vehicle, at-RA or 4-oxo-RA mice were sorted, respectively, in at least 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 was conducted using the Arcturus PicoPure RNA Isolation Kit (Applied Biosystems) following the manufacturer's guidelines. DNAse treatment was carried out employing 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, 12-14 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.