RNA-seq Illumina prepared with 10X 3' of homo sapiens: PBMCs
Library construction was performed using an automated workflow. The cDNA was fragmented and repaired with the addition of an A-tail by incubating at 32C for 5 minutes and 65C for 30 minutes, using the Chromium Single Cell 3 Library Construction Kit v3.1 (10x Genomics, PN-2000160). A 0.8 double-sided SPRI bead cleanup (Beckman Coulter) was performed to remove undesired fragments. Adapters were ligated onto the DNA at 20C for 15 minutes, followed by another 0.8 SPRI bead cleanup. Dual-indexed barcodes (10x Genomics, 1000215) were added, and libraries were amplified using PCR with the following conditions: initial denaturation at 98C for 45 seconds, followed by 12 cycles of 98C for 20 seconds (denaturation), 54C for 30 seconds (annealing), and 72C for 20 seconds (extension). Libraries underwent a final 0.8 double-sided SPRI bead cleanup for size selection. The quality of the final libraries was assessed using Quant-iT PicoGreen (Thermo Fisher Scientific) and an Agilent TapeStation (Agilent) to confirm the expected library size. Libraries were pooled to achieve the desired number of reads per cell for downstream analysis. Libraries were sequenced using NovaSeqX (28-bp Read 1, 10-bp i7 index, 10-bp i5 index, and 90-bp Read 2).