GSM9316593: WT CD45+, normal lung; Mus musculus; RNA-Seq
The isolated lung tumors from WT and Rab37 KO mice were minced into small fragments (~1 mm³) in ice-cold HBSS and resuspended in digestion buffer (HBSS with Mg²⁺/Ca²⁺ supplemented with 2 mg/ml collagenase type I and 40 U/ml Dispase I). Samples were incubated at 37 °C for 30 min with gentle agitation. The digested tissue was passed through a 70-µm cell strainer, washed and resuspended in PBS, and red blood cells were removed using RBC lysis buffer when necessary. Cells were then stained with a fixable Viability Dye and fluorochrome-conjugated anti-CD45 antibody for 20 min at 4 °C, washed, and resuspended in FACS buffer (HBSS without Ca²⁺/Mg²⁺, 2% v/v FCS, 5 mM EDTA). Live CD45⁺ cells were sorted from WT and Rab37 KO normal lung tissue or tumors on a FACS  using unstained and fluorescence-minus-one controls from C57BL/6 mice as negative controls. Sorted CD45⁺ cells were washed in HBSS-/- and finally resuspended in HBSS-/- supplemented with 0.04% w/v BSA at an appropriate concentration for single-cell RNA-seq library preparation. Single-cell RNA-seq libraries were generated using the Chromium Single Cell 3′ Reagent Kits v2 (10x Genomics) according to the manufacturer's instructions. Briefly, sorted live CD45⁺ cells from WT and Rab37 KO lung tumors were resuspended in the 10x master mix and loaded onto a Chromium chip together with partitioning oil and barcoded gel beads to produce gel bead-in-emulsions (GEMs). Within each GEM, cell lysis occurred and poly-A RNA was reverse-transcribed into cDNA carrying an Illumina Read 1 (R1) primer sequence, a Unique Molecular Identifier (UMI), and a 10x cell barcode. Barcoded cDNA from all GEMs was then pooled, cleaned up with Silane DynaBeads, and amplified by PCR. Appropriately sized cDNA fragments were selected using SPRIselect reagent and used for library construction. During library preparation, Illumina Read 2 (R2) primer sequence, paired-end constructs containing P5 and P7 adapter sequences, and a sample index were added to generate the final sequencing-ready libraries.