GSM6601928: HaHi02_CITE; Homo sapiens; RNA-Seq
Cryopreserved PBMC were thawed, stained with live/dead stain, and anti-human CD56, CD19, CD3, CD14 and CD16 antibodies, and TotalSeqTM-C oligonucleotide-conjugated antibodies (Biolegend, one different per sample).CD14+ monocytes were defined as live CD3-CD19-CD56-CD14+ cells. For each sample, 15,000 CD14+CD16+ and 15,000 CD14+CD16- monocytes were sorted using BD Aria 3 cell sorter into low-retention 1.5-ml collection tubes (Thermo Fisher Scientific), containing 0.5 ml of a 1:1 solution of phosphate-buffered saline (PBS):FBS supplemented with ribonuclease inhibitor (1:100; Takara Bio), and pooled together. Following cell sorting, cells were washed with ice-cold PBS, centrifuged for 10 min (600g at 4°C), gently resuspended in ice-cold PBS supplemented with 0.04% ultrapure bovine serum albumin (Sigma-Aldrich). The library preparation was performed using a 10x Genomics 5′ Tag v2 chemistry kit with dual indexes and feature barcoding technology for cell surface proteins. The amplification of complementary DNA was carried out with 13 cycles of amplification; the 5′ Tag gene expression libraries and the corresponding hashtag libraries were generated separately with 13 and 8 cycles of amplification, respectively. The libraries were sequenced using the Illumina NovaSeq 6000 sequencing platform with the following read lengths: read 1, 101 cycles; read 2, 101 cycles; i7 index, 10 cycles; i5 index, 10 cycles.