GSM9130865: GFPlo non-cardiomyocyte cells_MI 7d; Mus musculus; RNA-Seq
In p16-CreER;R26-mTmG mice subjected to MI surgery, cardiac P16-positive cells were lineage-traced by administering tamoxifen via oral gavage on day 5 post-MI. At day 7, the infarcted region of the heart was dissected, enzymatically digested by the Langendorff perfusion method. The resulting cell suspension was filtered through a 70-μm cell strainer and centrifuged at 50 g for 5 minutes to pellet and remove cardiomyocytes. The supernatant was collected and centrifuged at 500 g for 5 minutes to obtain a cell pellet depleted of cardiomyocytes. Erythrocytes were subsequently subjected to lysis, followed by fluorescence-activated cell sorting (FACS) to isolate distinct populations: GFPhi and GFPlo cell populations. After cell sorting, cell viability and number were assessed using the Countstar Rigel S2 fluorescence analyzer and AO/PI staining. Subsequently, cell debris and non-viable cells were removed using Miltenyi kits (130-109-398/130-090-101). The resulting live cells were washed twice with RPMI 1640 and resuspended at 1×10e6 cells/mL in 1×PBS containing 0.04% BSA. scRNA-seq libraries were prepared according to the manufacturer's instructions of Beijing SeekGene BioSciences Co., Ltd (Beijing, China).  Single-cell RNA-Seq libraries were prepared using SeekOne®Digital Droplet Single Cell 3'library preparation kit (SeekGene). Briefly, appropriate number of cells were mixed with reverse transcription reagent and then added to the sample well in SeckOne®DD Chip S3. Subsequently Barcoded Hydrogel Beads (BHBs) and partitioning oil were dispensed into corresponding wells separately in chip S3. After emulsion droplet generation reverse transcription were performed at 42℃ for 90 minutes and inactivated at 80℃ for 15 minutes. Next, cDNA was purified from broken droplet and amplified in PCR reaction. The amplified cDNA product was then cleaned, fragmented, end repaired，A-tailed and ligated to sequencing adaptor. Finally the indexed PCR were performed to amplified the DNA representing 3' polyA part of expressing genes which also contained Cell Barcode and Unique Molecular Index. The indexed sequencing libraries were cleanup with SPRl beads, quantified by quantitative PCR (KAPA Biosystems KK4824) and then sequenced on illumina NovaSeq 6000 with PE150 read length.