title: GSM9234730: Batch 1, pool 3, GEX; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen PBMC were thawed and prepped according to 10x genomics protocol. Cells were stained for viability with Live/Dead Near-IR stain (ThermoScientific, Waltham, MA) and sorted using a Cytek Aurora CS cell sorter (Bethesda, MD). Individual subject live cell suspensions were blocked using 5 µl of human TruStain FcX Fc Blocking reagent (BioLegend, 422302). Subsequently, they were incubated with 1 µl (0.5 µg) of the respective TotalSeq-B anti-human Hashtag antibodies 1–4 (BioLegend) for 30 min at 4 °C. After staining, the samples were washed and pooled into a single tube and counted for viability The scRNA-seq libraries were prepared using the Chromium Single-Cell 3′ Library and Gel Bead Kit v3.1 (10x Genomics, PN-1000268). 20,000 TotalSeq antibody-stained live cells were suspended in PBS (Gibco, 14190-144) with 0.04% bovine serum albumin (BSA; Sigma-Aldrich, A7030) buffer and loaded onto the Chromium Next GEM chip G, where cells and barcoded beads were partitioned into beads-in-emulsion (GEMs). GEMs were lysed, followed by reverse transcription of the released mRNA and isolation and amplification of the barcoded complementary DNA by PCR. Subsequent separation of hashtag/surface protein oligo-derived cDNAs (<200 bp) and mRNA-derived cDNAs (>300 bp) was accomplished using 0.6× SPRI bead (Beckman Coulter) purification on cDNA reactions