GSM8864980: MCF-7 NTC knockout_48 hours hypoxia; Homo sapiens; RNA-Seq
Cells were extracted and prepared for sequencing according to HIVE kit producer Honeycomb Biotechnologies. Cells were treated for 48 hours in normoxia or hypoxia, and afterward, they were prepared as single-cell suspensions with the use of 0.5mg/ml porcine trypsin in 0.5 mM EDTA (pH 8.0).  A total of 20,000 cells were seeded under the same oxygen conditions on HIVE Collectors (Honeycomb Biotechnologies). The HIVE devices, allow individual cells to settle into picowells that contain barcoded mRNA-capture beads. Picowells were sealed to isolate each cell-bead pair, and cells were lysed to release mRNA, which then binds to the barcoded beads. Beads were extracted from the HIVE and the captured mRNA was amplified to generate sequencing-ready libraries. Pooling the libraries from multiple samples and performing the sequencing with custom sequencing primers. The libraries were prepared according to the Honeycomb Biotechnologies protocol. A lysis solution from the kit was used to release RNA from the cells, then a provided hybridization solution was used to facilitate mRNA capture onto barcoded beads within the HIVE. The hybridization solution was removed and barcoded beads that contained captured mRNA were retrieved, then the beads were transferred to a filter plate and washed using bead recovery solution. Afterward, the specific enzymes and buffers from the HIVE kit were used for the first-strand synthesis reaction - synthesizing complementary DNA (cDNA) from the captured mRNA. Then second-strand synthesis to generate double-stranded cDNA was performed. Next, the full-length cDNA was amplified using PCR (oligos and enzymes provided in the HIVE kit). With the use of SPRI magnetic beads amplified cDNA was purified. Next, unique index sequences to each sample were assigned using provided by the company indexed PCR primers, and the libraries were amplified. Then a final SPRI bead purification to remove unwanted PCR byproducts was done and the final sequencing-ready libraries were eluted. Next, library quality and concentration were checked using Qubit and fragment analysis tools.