title: GSM9293728: 4T1 pooled tumors, KD-AFD, combined arginine restriction by Ass1-knockdown and arginine-free diet; Mus musculus; RNA-Seq
library_construction_protocol: On day 16 post-inoculation, mice were sacrificed, and tumors were resected and weighed. Excised 4T1 tumors were mechanically and enzymatically dissociated using the gentleMACS system (Miltenyi) in RPMI medium containing collagenase A and DNase I at 37°C. The resulting single-cell suspensions were filtered and subjected to red blood cell lysis. Equal numbers of viable cells from 4–5 tumors per treatment group were pooled, and dead cells were removed using a Dead Cell Removal Kit (Miltenyi). Single-cell suspensions with viability above 70% were processed on a Chromium Controller (10x Genomics) to generate GEMs. Barcoded cDNA was synthesized, amplified, and used to prepare libraries with the Chromium Single Cell 3′ Library Kit. Libraries were quantified, pooled, and sequenced on an Illumina NovaSeq 6000 (SP 100 cycles), targeting ~50,000 reads per cell.