GSM8606017: Day 26 Human Heart Organoids; Homo sapiens; RNA-Seq
Culture medium was carefully removed and discarded from the hHO. The hHO was washed once with PBS to remove any loosely attached cells. RPMI+B27 with 2% BSA (w/v) was prepared and filtered to make sterile. Cardiomyocyte Dissociation Medium (STEMCELL) was warmed to 37°C prior to dissociation. 500µL of the Cardiomyocyte Dissociation Medium was applied to the organoid and shaken at 37˚C at 250rpm for 5 minutes. The dissociation medium was then collected from the organoid containing RPMI+B27 with 2% BSA. The dissociation medium addition, incubation, and collection was repeated until 20 minutes passed. The collection was filtered with a 40µm filter and then centrifuged at 300g for 5min. The supernatant was decanted, and the pelleted cells were resuspended in a smaller volume of FACS buffer for flow cytometry or RPMI+B27 + 2% BSA for scRNAseq. After the cells were resuspended, the cells were filtered again with a 40µm filter. Libraries were prepared using the 10x Chromium Next GEM Single Cell 3' Kit, v3.1 and associated components. Libraries were prepared using the 10x Chromium Next GEM Single Cell 3' Kit, v3.1 and associated components. Completed libraries were QC'd and quantified using a combination of Agilent 4200 TapeStation HS DNA1000 and Invitrogen Collibri Library Quantification qPCR assays. Libraries from each pool were pooled in equimolar proportions and the pool quantified again using the Invitrogen Collibri qPCR assay. These two project pools were then pooled in equal proportions and this final pool again quantified by qPCR. The pool was loaded onto 1 lane of an Illumina NovaSeq 6000 S4 flow cell (v1.5) and sequencing was performed in a 2x150bp paired end format using 300 NovaSeq v1.5 reagent cartridge. Base calling was done by Illumina Real Time Analysis (RTA) v3.4.4 and output of RTA was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.20.0. For Bcl2fastq only the first 28 bases of read #1 and 100 bases of read #2 were output. The first 28bp of read 1 includes the 10x cell barcodes and UMIs, read 2 is the cDNA read.