GSM9059697: WT_DOX_01; Mus musculus; RNA-Seq
Mouse hearts were minced and placed into a 15-ml conical tube containing DMEM with Collagenase I (450 U/ml), DNase I (60 U/ml), and hyaluronidase (60 U/ml). The mixture was then incubated at 37 ℃ for 30 minutes with agitation. Digestion was stopped by adding DMEM with 2% FBS, followed by passing the mixture through a cell strainer (40-mm) into a 50-ml conical tube. The filtrate was then centrifuged at 300g for 5 minutes at 4 ℃. After discarding the supernatant, the cell pellet was resuspended in 1 ml ACK lysing buffer and incubated at room temperature for 5 minutes. Subsequently, 9 ml DMEM was added, and the cell viability (~80%) was assessed. Batch1: Chromium Next GEM Chip G Single cell 3' Reagent V3.1 kit from 10× genomics, with 10,000 cells loaded into a Chip G for GEM generation. Reverse transcription, barcoding, complementary DNA amplification, and purification for library preparation were performed according to the Chromium 3' V3.1 protocol. Batch2: Library preparation was carried out using the Chromium X platform and the Fixed RNA Profiling 3' v3.1 chemistry, following the manufacturer's standard protocol.