GSM9177584: acaA- PkaCoe_04hr : replicate 1; Dictyostelium discoideum; RNA-Seq
Single-cell suspensions were immediately loaded into the 10x Genomics Chromium X system and processed according to the manufacturer's recommended protocol. Libraries were prepared using the Chromium Single Cell Gene Expression 3' v3.1 or v4 kits (10x Genomics). Briefly, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded onto a Chromium X instrument (10x Genomics) to generate Gel Beads-in-Emulsions (GEMs). Within each GEM, full-length cDNA was synthesized, incorporating an Illumina R1 primer sequence, a cell-specific barcode, and a Unique Molecular Identifier (UMI). Following GEM disruption, cDNA from individual cells was pooled, purified using Dynabeads MyOne Silane Beads (Thermo Fisher Scientific), and amplified by PCR. The amplified products were then fragmented to optimal sizes, followed by end-repair, A-tailing, and adaptor ligation to add the Illumina R2 primer sequence. Final libraries were generated through a second round of amplification, during which paired-end constructs with P5 and P7 sequences were added.