GSM8676670: EB, 7th day, RNA; Homo sapiens; RNA-Seq
***HSPC and macrophage*** Cells were washed in 1 x PBS-/- supplemented with 0.04% BSA and sorted using the BigFoot cell sorter (Thermo Fisher) on days 7 or 14. For in vitro-differentiated adherent cells, cells were lifted using enzyme-free cell dissociation buffer (ThermoFisher) before washing and sorting. Nuclei were isolated following the 'Low Cell Input Nuclei Isolation' sub-protocol and immediately processed using the Chromium Next GEM Single Cell Multiome + Gene Expression kit. ***EB*** Flash-frozen D7 EBs were thawed on ice. Nuclei isolation was performed according to the 10X Genomics demonstrated protocol 'Nuclei Isolation from Complex Tissues for Single Cell Multiome ATAC + Gene Expression Sequencing' (CG000375 Rev B). Briefly, EBs were homogenized using a 1000 ul wide-bore tip in lysis buffer and incubated on ice for 5 minutes. Cells were then filtered through a 70 um strainer and centrifuged at 500g for 5 min at 4C. The supernatant was removed and 1 ml of PBS + 1% BSA + 1U/ul RNase inhibitor (Roche; Cat. No. 3335402001) was added and incubated on ice for 5 minutes. The pellet was resuspended using a wide bore tip and centrifuged at 500g for 5 minutes at 4C. The pellet was then resuspended in 100 ul 0.1X lysis buffer and incubated on ice for 2 minutes. 1 ml of buffer was added to mix the pellet and centrifuged at 500 g for 5 minutes at 4C. The supernatant was removed and resuspended in chilled diluted nuclei buffer (10X Genomics). The nuclei were diluted to 3000-8000 nuclei/ul by using a Countess 3 Automated Cell Counter and proceeded to Chromium Next GEM Single Cell Multiome ATAC + Gene Expression. ***HSPC and macrophage*** Single Cell Multiome ATAC and Gene Expression libraries were prepared according to the 10X Chromium Next GEM Single Cell Multiome ATAC + Gene Expression user guide for standard Illumina paired-end sequencing. ***EB*** Single nuclei libraries were generated using the 10X Genomics Chromium Controller following the manufacturer's protocol for Multiome (ATAC+GEX) analysis. In brief, transposase was added to the nuclei suspension and incubated, before adding barcode and RT master mix and loading into the Chromium Controller chip with appropriate gel beads. Following the generation of single-cell gell bead-in-emulsions (GEMs), reverse transcription of the RNA and barcoding of the fragmented DNA was performed. The resulting product was cleaned and both the cDNA and barcoded, fragmented DNA were amplified. One fraction of this product made the ATAC library via indexing PCR. The other fraction was amplified again to isolate the cDNA further, which was then quantified by Qubit (Invitrogen) and assessed for size on the TapeStation 4200 (Agilent). It underwent enzymatic fragmentation, size selection, adapter ligation, and indexing PCR. Final library quality was assessed using the LabChip GX Touch HT(Revvity) and was quantified by Qubit. Pooled libraries were then subjected to paired-end sequencing according to the manufacturer's protocol (Illumina NovaSeq6000). BclConvert software (Illumina) was used to generate de-multiplexed Fastq files and the CellRanger Pipeline (10X Genomics) was used to align reads and generated count matrices. OTHER: Multiome (RNA and ATAC)