GSM9060554: P3 Pc pool2 6% ficoll layer (primary asci); Pneumocystis carinii; RNA-Seq
single-cell RNA sequencing (scRNA-seq) was performed using the Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index) from 10x Genomics. To facilitate the lysis of P. carinii asci, which possesses a robust β-1,3-D-glucan cell wall, 1 µL of Zymolyase 100T (10 mg/mL; Asmbio, Cambridge, MA) was added to the gel bead mix before droplet generation. This enzymatic treatment enhances cell wall digestion, thereby improving the efficiency of in-droplet lysis during the scRNA-seq workflow. Briefly, cells were resuspended in the master mix and loaded into the Chromium chip with partitioning oil and gel beads to generate gel bead-in-emulsions (GEMs). Polyadenylated RNA underwent reverse transcription within each GEM, incorporating an Illumina TruSeq Read one primer sequence, a Unique Molecular Identifier (UMI), and a 10x Barcode. Post GEM-recovery, barcoded cDNA was purified using Silane DynaBeads and amplified via 14 cycles of PCR. The amplified cDNA was then enzymatically fragmented, size-selected, adapter-ligated, and subjected to sample index PCR to construct sequencing libraries. Libraries were pooled and sequenced on an Illumina NovaSeq 6000 platform using an S4 flow cell with the following parameters: Read 1: 28 cycles; i7 Index: 10 cycles; i5 Index: 10 cycles; Read 2: 90 cycles.