GSM9091060: 4T1,lymph node,control; Mus musculus; RNA-Seq
Tumors and draining lymph nodes (dLNs) were harvested and processed to generate single-cell suspensions. Tumor tissues were finely minced and enzymatically digested using Collagenase D (Roche, Cat# 11088882001) for 40 minutes at 37 °C with agitation at 250 rpm. dLNs were mechanically dissociated by gently mincing between sterile glass slides. Following dissociation, tissues were passed through a 70 μm cell strainer to remove debris and obtain a single-cell suspension. The cell suspensions were centrifuged, resuspended in completed media, and filtered again through a 30 μm cell strainer. For tumor samples, the single-cell suspension was layered over Histopaque-1077 (Sigma-Aldrich, Cat# 10771) in a 50 mL conical tube and centrifuged at 400 × g for 30 minutes at room temperature with acceleration and brake set to level 1. The upper plasma layer was carefully removed and discarded, and the opaque interface was collected into a fresh conical tube. Collected cells were washed by adding 10 mL of complete media (RPMI-1640 supplemented with 10% FBS, 1% penicillin-streptomycin, NSAA, Sodium pyruvate and β-Me), followed by centrifugation at 250 × g for 10 minutes. This wash step was repeated twice. Final cell pellets were resuspended in 5 mL of complete media and counted using a hemocytometer. For tumors and dLNs, CD8 T cells were enriched using the CD8 (TIL) Isolation Kit (Miltenyi Biotec, Cat# 130-116-478) according to the manufacturer's instructions. Live CD8+ T cells from TDLN day 5 post treatment were sorted for single-cell RNA-seq:. Sorted cells were then loaded onto a 10x Chromium Chip A using reagents from the Chromium Single-Cell 5' Library and Gel Bead Kit (10x Genomics) according to the manufacturer's protocol. Amplified cDNA was used for both 5' RNA-seq library generation and TCR V(D)J targeted enrichment using the Chromium Single-Cell V(D) J Enrichment Kit for Mouse T Cells (10x Genomics). 5' RNA-seq and TCR V(D)J libraries were prepared following the manufacturer's user guide (10x Genomics). The final libraries were profiled using the Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies) and quantified using the Kapa Library Quantification Kit (Kapa Biosystems)