GSM5396335: scHTO_CD4+CD45RO+_lane 2; Homo sapiens; OTHER
Single nuclei were stained and loaded onto a 10x Genomics Chromium controller for generation of NEAT-seq libraries Antibody-stained cells in 1X Nuclei buffer were processed using the 10X Genomics Multiome kit as indicated in the standard protocol (Rev A) to generate ATAC-seq and RNA-seq libraries. During the pre-amplification step, Truseq read 2 (CAGACGTGTGCTCTTCCGATC) and Nextera read 2 (GGCTCGGAGATGTGTATAAGAGACAG) primers were spiked in at 0.2uM final concentration to amplify ADT and HTO oligos. To generate ADT and HTO libraries, 35ul of pre-amplification product from step 4.3p was amplified with indexing primers using 2X NEB Next High-Fidelity PCR Master Mix (M0541). A double-sided SPRI bead clean up was performed using 0.6X SPRI beads (retaining supernatant) and then adding additional SPRI beads to a final concentration of 1.2X, washing with 80% ethanol, and eluting ADT or HTO libraries from beads using EB buffer.