GSM7033735: MEF all remaining barcodes [scRNA-seq]; Mus musculus; RNA-Seq
Cells were harvested at days 2, 7, or 14. Cultures dissociated into single cells after two PBS washes by Accutase digestion. Cells were lifted using cell scrapers, then suspensions were then passed through 70 μm filters into 9 mL of pre-warmed MEF medium. Cells were pelleted (800 RPM, 5 min) and resuspended in 1 mL HBSS + 0.04% BSA to > 3.5 million cells/mL, then fixed through the addition of 4× volume of ice-cold methanol to a final concentration of 80% methanol. Cells were then stored at -20° C. Barcoding oligonucleotides for multiplexing were designed based upon the ClickTag scheme and ordered with a 5' amine group label. A methyltetrazine group was conjugated to the oligos via the 5'-amine group. Membrane proteins on the fixed cells were conjugated to an amine-trans-cyclooctene group. Subsequent incubation of oligos and cells according to the ClickTag protocol allowed chemical labeling of cells. ~10,000 cells were multiplexed and loaded per GEM well in the Chromium Controller (10x Genomics), and the Chromium Single Cell 3' v2 reagent kit was used according to the manufacturer's instructions for gene expression library generation. We performed modifications at the cDNA and library preparation steps as suggested in the ClickTag protocol to generate barcoded oligonucleotide libraries in parallel. Gene expression and barcode libraries were then diluted to equimolar amounts, pooled at a 9 to 1 ratio, and 26 + 98 bp paired-end sequencing was performed using NovaSeq 6000 platform (Illumina).