GSM5593390: Spin-EB D3 - Donor 2; Homo sapiens; RNA-Seq
For Single-Cell RNA-Seq samples, colonies harvested at day 5, the cell culture medium was aspirated, 800 µL of Accutase were added on top of the cells, and they were incubated for 5 minutes at 37 °C After that, 1 mL of pre-warmed KO-DMEM were added to each sample and the cell were pipetted several time to create single cell suspension. Cells were then transferred to 15-mL falcon tubes. For processing of 3D aggregates, they were collected at day 8 from cell seeding into15 mL falcon tube and centrifuged at 100 g for 3 minutes. The supernatant was aspirated, and 1 mL of pre-warmed Accutase were added to each falcon tube for 9 mins at 37 °C. The aggregates were pipetted up and down several time to create single cell suspension. Falcons containing single cells were centrifuged again at 1000 RPM for 4 minutes. Then, they were re-suspended in 1 mL of PBS + 1 µL Y-27632 ROCK Inhibitor + 0.04% BSA. The suspension were filtered through 40 µm cell strainer to remove cell clumps. The cells were counted and centrifuged again, and the cells density was adjusted to 500-1000 cells/µL. Cells were kept on ice until further processing. For RNA-Seq experiment, self-detached aggregates or Spin-EBs were incubated in EB-Medium containing 10% FCS for 7 days. Samples were isolated at day 0, 3, and 7. Total RNA were isolated using Nucleospin RNA kit (Macherey-Nagel), Quality control was done with a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, USA). RNA was stored at -80 until further processing. For single-cell RNA-Seq, The cell suspension was processed by the chromium platform (V3) from 10X Genomics according to the manufacturer's 3' protocol to obtain a sequencing library. The cells were deposited into the microfluidic platform provided by 10X Genomics. Chromium controller was used to partition cells into Gel Bead-In-EMulsions (GEMs). Cell lysis was carried out in the GEMs, which included a unique barcode for each bead. The Read1 was added during the GEMs lysis and cDNA construction. The barcoded cDNA was then purified using Dynabeads and amplified using PCR and random primers. Enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and Read 2 primers are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification. The library was then sequenced using Nextseq 550 to get paired-ended reads. For Bulk-RNA-seq experiment, the library preparation was carried out using QuantSeq 3´-mRNA (Lexgon) according to the manufacturer instruction. Sequencing was done using NovaSeq 6000 sequencer (Ilumina). Library preparation and sequencing was carried out by Life&Brain (Bonn, Germany).