title: GSM8911928: Samples 25 to 32 from cmo feature table associated to CR02_G_GEX_L003; Homo sapiens; RNA-Seq
library_construction_protocol: For each sample, live cells were transferred to 2 mL DNA LoBind tube  in mL PBS/0.04% w/v BSA and centrifuged at room temperature at 300×g for 5 min. The pellets were resuspended in 100 μL of a distinct Cell Multiplexing Oligo (CMO; 3' CellPlex Kit Set A, PN-1000261, 10X Genomics; Pleasanton, CA). After 5 min of incubation at room temperature, cells washed 3 times with 2 mL ice-cold PBS/1% w/v BSA by centrifuging at 300×g for 5 min at 4 °C, then incubated with 10% v/v 7-aminoactinomycin D (7-AAD) in 200 µL PBS/2% w/v BSA for 10 min. 5×105 7-AAD+(live) single cells per sample were sorted with Sony SH800S Cell Sorter (Sony Biotechnology, San Jose, CA) in sterile tubes kept at 4°C, containing 100 µL RPMI + 0.2 U/mL RNAse inhibitor (ThermoFisher Scientific, Waltham, MA). Samples were subsequently pooled in a 2 mL DNA LoBind tube, pelleted at 300×g for 5 min at 4°C, and diluted to a final concentration of 1.5×106 cells/mL. 49,500 cells were loaded in a single channel of a Chromium Next GEM Chip G to prepare scRNA-Seq libraries using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 and the Chromium X controller (all from 10X Genomics, Pleasanton, CA), following the manufacturer's instructions to prepare gene expression (GEX) and CMO libraries (protocol CG000388 Rev B). GEMs were broken and cDNAs were purified, pre-amplified by 11 cycles of PCR, and quality controlled using an Agilent D5000 ScreenTape and an Agilent 2150 Tapestation (Agilent, Santa Clara, CA). 25% of each cDNA was fragmented, end-repaired, A-tailed, ligated to Illumina adapters, and amplified by 12 cycles of PCR using an individual primer pair from the Dual Index Kit TT, Set A (10X Genomics) to generate GEX libraries. CMO libraries were generated using the Dual Index Kit NN, Set A (10X Genomics), quality controlled using an Agilent D1000 ScreenTape and an Agilent 2150 Tapestation, quantified using the Qubit dsDNA HS Assay (Invitrogen), and pooled at 6:1 molar ratio for GEX:CMO. The library pool was sequenced using an Illumina NovaSeq 6000 (Illumina, San Diego, CA) flow cell, performing 28 cycles for read 1, 90 cycles for read 20, and 10 cycles for each index.