Gemcode single cell 3 gel bead and library v2 kit

Single-cell sequencing for the tabula muris project was previously described [17 (link)]. In brief, single cells were captured in droplet emulsions using the GemCode Single-Cell Instrument (10x Genomics), and scRNA-seq libraries were constructed as per the 10x Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V2 Kit. The samples were diluted in PBS with 2% FBS to a concentration of 1.000 cells per μL. Cells were loaded in each channel with a target output of 5.000 cells per sample. All reactions were performed in the BioRad C1000 Touch Thermal cycler with 96-Deep Well Reaction Module. Amplified cDNA and final libraries were evaluated on a Fragment Analyzer using a High Sensitivity NGS Analysis Kit (Advanced Analytical). Equal volumes of 16 libraries were pooled for sequencing on the NovaSeq 6000 Sequencing System (Illumina).
Sequencing of Fzt:DU and C57BL/6NRj samples were conducted by Genewiz (GENEWIZ Germany GmbH, Leipzig, Germany). Similar to the tabula muris project, single nuclei were captured in droplet emulsions on the 10xGenomics system and sequenced on the NovaSeq 6000 Sequencing System (Illumina, San Diego, CA, USA). In contrast to the tabula muris sequencing, cells were loaded with a target output of 10,000 cells per sample and the snRNA-seq libraries were constructed using Library V3 chemistry.

Tumor samples were collected and transported in IMDM (12440053; Gibco): RPMI-1640 (A1049101; Gibco) 4:1 medium on ice. Single-cell suspensions were obtained from tumor biopsies through mechanical and enzymatic dissociation. Then the samples were washed with phosphate buffered saline (PBS; ThermoFisher Scientific). Viability was confirmed to be > 70% in all samples using trypan blue (15250061; ThermoFisher Scientific). Cell suspensions were filtered using a 70 μm filter (431751; Falcon), and dissociated cells were pelleted and resuspended in PBS with 1% bovine serum albumin (BSA; Sigma-Aldrich). Single cells were captured in droplet emulsions using the GemCode Single-Cell Instrument (10× Genomics), and scRNA-seq libraries were constructed per the 10× Genomics protocol using GemCode Single-Cell 3′ Gel Bead and Library V2 Kit. All libraries were sequenced on the NovaSeq 6000 Sequencing System (Illumina).

Single cells were captured in droplet emulsions using a GemCode Single-Cell Instrument (10X Genomics), and scRNA-seq libraries were constructed with the 10X Genomics protocol using a GemCode Single-Cell 3′ Gel Bead and Library V2 Kit. The libraries were sequenced using a HiSeq X-10 Sequencing System (Illumina). scATAC-seq experiments were performed as previously described [15 (link)].