title: GSM6098652: ADRA03_IOF3_4_A [ADT-derived cDNA]; Homo sapiens; OTHER
library_construction_protocol: Sample pool was counted and loaded on five lanes of the 10x Genomics NextGem 3'v3.0 assay as per the manufacturer's protocol with a targeted cell recovery of 30,000 cells per lane. Gene expression and ADT libraries were made as per the 10x Genomics demonstrated protocol (https://assets.ctfassets.net/an68im79xiti/40xc58fJNcCWipI1YIdXoS/a87092c1c1faa0f316c552a97229a268/CG000185_ChromiumSingleCell3_v3_FeatureBarcoding_CellSurfaceProtein_UG_Rev_D.pdf). Hashtag oligonucleotides (HTO) were enriched during cDNA amplification with the addition of 3 pmol of a HTO Additive primer (5'GTGACTGGAGTTCAGACGTGTGCTC). This PCR product was isolated from the mRNA-derived cDNA via SPRISelect size selection, and libraries were made as per the New York Genome Center Hashing protocol. The hashed samples were then pooled and stained together with a cocktail of 73 Biolegend Totalseq-A antibodies. All libraries were quantified via Agilent 2100 hsDNA Bioanalyzer and KAPA library quantification kit (Roche Cat# 0796014001.) Gene expression libraries were sequenced at a targeted depth of 25,000 reads per cells. ADT libraries were sequenced at a targeted read depth of 10,000 reads per cell. HTO libraries were sequenced at a targeted read depth of 1,000 reads per cell. Libraries were sequenced on the Illumina NextSeq Hi 500/550 75 cycle kit, NovaSeq 6000 100 cycle kit, or NovaSeq S2 100 cycle kit with run parameters set to 28x8x0x60 (R1xi7xi5xR2). Hashtag Antibody Staining: Cells were thawed and counted using the Nexcelom Cellaca. An aliquot of 500,000 cells from each sample were centrifuged at 350 RCF for 5 minutes (4°C). The supernatant was discarded and the pellets were resuspended in a combinatorial hashtag antibody solution (see: Table1) and incubated at 4°C for 20 minutes. Hashtag antibodies were conjugated as per New York Genome Center hashing protocol (https://citeseq.files.wordpress.com/2019/02/cite-seq_and_hashing_protocol_190213.pdf). Stained cells were washed three times in 1mL wash buffer (PBS + 0.5% BSA) by 4°C centrifugation at 350 RCF to remove unbound antibodies. Washed cells were resuspended in 150ul wash buffer and counted using a Nexcelom Cellaca. Hashed samples were pooled (see: Table2) and centrifuged at 350 RCF for 5 minutes (4°C). Supernatant was removed and pellet was resuspended in 100ul of antibody cocktail (see: Table3) and incubated at 4°C for 30 minutes. Stained cells were washed three times in 1mL PBS + 0.5% BSA by 4°C centrifugation at 350 RCF to remove unbound antibodies. The washed cells were resuspended in wash buffer to achieve a target concentration of 4 million cells/mL.