title: Single Cell RNA-seq of gallus gallus embryonic tissue
design: The scRNA-seq library was generated following the 10x Genomics protocol designed for the Chromium Next GEM Single Cell 3 Reagent Kits v3. Briefly, the Chromium Next GEM Chip G was loaded with a mixture of the final cell suspension and Master Mix (RT Reagent B 2000165, Template Switch Oligo 3000228, Reducing Agent B 2000087, and RT Enzyme C 2000085/2000102). The remaining rows were allocated for Single Cell 3 v3.1 Gel Beads (2000164) and partitioning oil, with one row left unfilled. Any unused wells were filled with a 50% glycerol solution, in line with the 10x Genomics protocol. The droplet formation was visually assessed according to the manufacturer's protocol. Upon the conclusion of the run, the GEMs were recovered from the chip and placed into a PCR tube on ice. The GEMs were subsequently incubated in a thermal cycler lasting 55 minutes (53C for 45 seconds, followed by 85C for 5 seconds). The GEMs cleanup procedure was performed according to the manufacturer's instructions similarly to what was previously described for scATAC-seq. Post-cleanup, and a recovered sample volume of 35l was combined with the cDNA Amplification Reaction Mix. An additional round of incubation was conducted, as following the settings described in the cDNA Amplification process outlined in the manufacturer s 10x Genomics protocol, for a total of 13 cycles. Finally, the sample underwent cleanup using SPRIselect (Beckman Coulter SPRIselect Reagent Kit, B23318), after which the quantification of cDNA was assessed using Invitrogen Qubit dsDNA assay-kits and a Qubit fluorometer (Thermo Fisher Scientific, 10616763).
identifiers.namespace: SUB14694584
identifiers.id: HH22_FEZ_RNA