GSM8959920: Multiplex of heart cells from eoEAM model; Mus musculus; RNA-Seq
The heart single cell suspensions were prepared as per 10X genomics single cell protocol (CG00053 Rev C). Briefly, the hearts were cut into halves using 4-chamber cut, and half a heart was saved for histology, while the other half for preparation of single cell suspension. Heart tissue was minced, followed by enzymatic digestion (2.2mg/mL Collagenase IV and 1.5mg/mL Dispase II) at 37°C for 45 minutes. Subsequently, samples were filtered through a 40µm filter, and red blood cells were lysed (eBioscience RBC Lysis Buffer). Cells were then resuspended in RPMI supplemented with 10%FBS and filtered through 30 µm MACS cell strainer. The cells were counted to determine concentration and viability using a hemocytometer after which they were subsequently fixed for chromium fixed RNA profiling. The fixation was done following 10X genomics Fixation of Cells & Nuclei for Chromium Fixed RNA Profiling protocol (CG000478 | Rev C). Briefly, the heart single cell suspensions were centrifuged at 400g and resuspended in Fixation Buffer, followed by storage at 4°C for 16-24 hours. Following a spin at 850g for 5 minutes at room temperature (22°C), the sample pellet is resuspended in chilled Quenching Buffer, and cell concentration is determined. Pre-warmed Enhancer and glycerol are added prior to storing cells -80°C. Library construction was performed using the Chromium Fixed RNA Profiling Reagent Kit (10x Genomics) according to the manufacturer's protocol (User Guide CG000527). Fixed cells were hybridized overnight with mouse transcriptome probe pairs and labeled with CMOs (BC001–BC004). Following CMO-based barcoding, cells were pooled and subjected to GEM (gel bead-in-emulsion) generation using the Chromium X instrument. Within each GEM, gel beads dissolved to release barcoded primers, and fixed cells lysed to expose hybridized RNA. Probe ligation and incorporation of barcoding primers occurred within GEMs. The primers included Illumina Read 1 (Read 1T), a 16 bp 10x GEM barcode, a 12 bp unique molecular identifier (UMI), and Capture Sequence 1. After GEMs were broken, Silane DynaBeads were used for cleanup, followed by pre-amplification and SPRIselect size selection. During library construction, P5 and P7 sequences, Illumina sample indices (i5, i7), and Read 2 (Read 2S) adapters were added to generate sequencing-ready libraries. Libraries were sequenced on a NovaSeq X Plus (10B flow cell) with the following read configuration: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.