Single cell 3 chip kit v2

To account for interindividual variability, we harvested pooled fresh tumor-bearing lungs from two mice in two independent cohorts of RPP orthotopic mice which were treated with control, YKL-5–124, anti-PD-1 and combination treatment for seven days. Single cell suspensions were achieved as described above and were sorted using DAPI staining. Cells were then resuspended into single cells with 0.4% BSA for 10x genomics processing. Cell suspensions were loaded onto a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Approximately 5,000 to 10,000 cells were loaded per channel. scRNA-seq libraries were prepared using the following Single Cell 3′ Reagent Kits: Chromium™ Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10x Genomics, Pleasanton, CA, USA) as previously described (Zheng et al., 2017 (link)), and following the Single Cell 3′ Reagent Kits v2 User Guide (Manual Part # CG00052 Rev A). Libraries were run on an Illumina HiSeq 4000 system (SY-401–4001, Illumina) as 2 × 150 paired-end reads, one full lane per sample, for approximately >90% sequencing saturation.

Approximately 10,000 CD45+ cells were loaded per channel on a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). scRNA-Seq libraries were prepared using the following Single Cell 3’ Reagent Kits v2: Chromium™ Single Cell 3’ Library & Gel Bead Kit v2, Single Cell 3’ Chip Kit v2, and i7 Multiplex Kit (catalog# PN-120237, PN-120236, # PN-120262, 10x Genomics)¹⁹ and following the Single Cell 3’ Reagent Kits v2 User Guide (Manual Part # CG00052), Rev A. Libraries were run on Illumina HiSeq 4000 as 2×150 paired-end reads, one full lane per sample, for approximately >90% sequencing saturation. There were some differences in downstream cell recovery, especially for inflamed samples for which fewer biopsies were obtained. Samples that failed to meet a minimum of 1,000 sequenced cells were deemed poor quality and removed from subsequent analysis. Quantitative analysis was performed in R and discussed in detail in the supplementary methods. The processed single cell count tables are provided in Gene Expression Omnibus; GSE162335. Raw sequence data are not public and are protected by controlled-access for patient privacy.

Kelly cells (sensitive, intermediate and resistant states) were grown to 70% confluence in T75 culture flasks. In brief, growth medium was aspirated and cells were treated with 0.25% Trypsin/EDTA for 3 min at 37 °C, after which cells were washed twice with 1× PBS. Cells were then resuspended into single cells at a concentration of 1 × 106 per ml in 1× PBS with 0.4% BSA for 10x Genomics processing. The sorted cell suspensions were loaded onto a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Approximately 5,000 cells were loaded per channel. scRNA-seq libraries were prepared using the following Single Cell 3′ Reagent Kits: Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10x Genomics) as previously described^{42 (link)}, and following the Single Cell 3′ Reagent Kits v2 User Guide (Manual Part CG00052 Rev A). Libraries were run on an Illumina HiSeq 4000 system (SY-401–4001, Illumina) as 2 × 150 pairedend reads, one full lane per sample, for approximately >90% sequencing saturation.