title: GSM8633707: KPH_post_depletion; Mus musculus; RNA-Seq
library_name: GSM8633707
library_construction_protocol: Individual macroscopic tumors were removed from the lungs and broken down into single cell suspensions. Pre-depletion cells were viably cryopreserved in 5% DMSO/FBS. The remaining cells were depleted of CD45-positive and CD31-positive cells using MACS with Miltenyi microbeads (CD45:130-052-301; CD31:130-097-418) and LD columns (130-042-901) following manufacturer recommendations. Post-depletion cells were viably cryopreserved in 5% DMSO/FBS. The day of library preparation, cells were thawed, washed in cold PBS, and resuspended in PBS + 0.5% BSA. Cells were stained with DAPI and sorted for single, live cells using a BD FACSAria cell sorter. Protocols used to generate scRNA-seq data with 10x Genomics Chromium platform can be found at https://support.10xgenomics.com/single-cell-gene-expression. In brief, the Chromium Single Cell Gene Expression Solution with 3' chemistry, version 3 (PN-1000075) was used to barcode individual cells with 16bp 10X barcodes and to tag cell specific transcript molecules with 10bp Unique Molecular Identifier (UMI) according to the manufacturer's instructions. The following protocol was performed at the High-Throughput Genomics Shared Resource at Huntsman Cancer Institute, University of Utah. Single cells were suspended in PBS with 0.04% BSA, and the cell suspension was passed through a 40 micron cell strainer. Viability and cell count were assessed on Countess II (Thermo Scientific). Suspensions were equilibrated to targeted cell recovery of 10,000 cells. 10x Gel Beads and reverse transcription reagents were added and cell suspensions were loaded to Chromium Single Cell A (PN-120236) to form Gel Beads-in emulsions (GEMs) - the nano-droplets. Within individual GEMs, cDNA generated from captured and barcoded mRNA was synthesized by reverse transcription at the setting of 53°C for 45 min followed by 85°C for 5 min. Subsequent A tailing, end repair, adaptor ligation and sample indexing were performed in bulk according to the manufacturer's instructions. The resulting barcoding libraries were qualified on Agilent D1000 ScreenTape on Agilent Technology 2200 TapeStation system and quantified by quantification PCR using KAPA Biosystems Library Quantification Kit for Illumina Platforms (KK4842). Multiple libraries were then normalized and sequenced on NovaSeq 6000 with 2 × 150 PE mode.