title: GSM8455168: CM_9w_RNA_XistBxC_+y_XY_3; Mus musculus; RNA-Seq
library_construction_protocol: Single cell suspensions of hearts were obtained by performing an enzymatic retrograde perfusion. To do so, the heart was harvested and cannulated at the aorta. Coronary arteries were flushed briefly with 1ml Perfusion Buffer. Afterwards, the cannula with the heart was attached to the perfusion pump system, and the heart was perfused with Perfusion Buffer for 1min (4ml/min). Next, the heart was rinsed with a Digestion Buffer for 10min (recirculating, 4ml/min) to enzymatically dissociate the ventricular cells. Then, the heart was detached, atria were discarded, and the ventricles were roughly homogenized in 2.5ml Digestion Buffer by cutting with scissors shearing with a 1ml syringe 30 times. After adding 2.5ml Stop Buffer, the syringe sheared the liquid again for 30 times. Ultimately, the suspension was filtered through a 100µm strainer and put on ice. To separate cardiomyocytes from non-cardiomyocytes, the suspension was centrifuged at 100xg at 4°C for 1min and supernatant was transferred. This was repeated once and the sediment, containing cardiomyocytes, was resuspended in 500µl TRIzol, homogenized using a pellet pestle. After adding another 500µl TRIzol the cardiomyocytes were frozen at -80°C for further use. The supernatant, containing non-cardiomyocytes, was spinned down at 400xg 4 °C for 7min. After discarding the supernatant, the pellet was resuspended in RBC-Lysis-Buffer at room temperature for 0.5min. Next, 2ml PBS was added, and suspension was centrifuged at the same settings, followed by the filtering through a 70µm strainer. Cells were blocked with Fc-block at 4°C for 15min, before using CD45-conjugated microbeads (Miltenyi) to bind CD45-positive cells. Afterwards, the cell suspension was separated into a CD45-positive and CD45-negative fraction using the autoMACS Pro Separator by Miltenyi. Then, both fractions were stained with Zombie Green to detect dead cells, followed by the macrophage staining for the CD45-positive fraction (CD45-positive, CD11b, CD64, F4/80) and the endothelial staining (CD45-negative, CD105) and the fibroblast staining (CD45-negative, CD140a) for the CD45-negative fraction. After washing twice, 2,000 cells of each cell type were sorted, immediately frozen on dry ice, and stored at -80°C for further use. After thawing, sorted MCL were resuspended in 1ml TRIzol and RNA was isolated according to manufacturer's instructions (Invitrogen, TRIzol Reagent, Cat. #15596018). Since the starting sample was small, 10μg of RNase-free glycogen was used as a carrier, and isopropanol precipitation was performed at -20°C overnight. RNA quality was measured on Agilent's TapeStation System. 0,5-3ng of RNA were used to generate RNA-seq libraries using Takara's SMART-Seq® v4 PLUS kit according to manufacturer's instructions. A quarter of reaction volume per sample was pipetted by the MANTIS liquid handler. Concentration and fragment distribution of completed RNA-seq libraries was measured on Agilent's TapeStation System.