GSM7890017: NCI-H226_CL3, NCI-H226, HTO; Homo sapiens; OTHER
The multiplexed single-cell Multiome (scMultiome) assay was performed following the TEA-seq 71 protocol with some modifications. Briefly, the cells were cultured and treated with different compounds in 6-well plates. Upon harvesting, the cells were trypsinized to single cell suspension, washed once by PBS. Samples from different conditions were labeled by the TotalSeq-A cell hashing antibodies with different hashtags (Biolegend) by incubating on ice for 20 minutes. After washing twice by PBS, the samples were pool together and permeabilized by the isotonic permeabilization buffer (20 mM Tris-HCl pH 7.4, Rnase Inhibitor 1 U/μL, 150 mM NaCl, 3 mM MgCl2, 0.01% digitonin) on ice for 15 minutes. Then the samples were washed with the isotonic buffer without digitonin, counted and immediately proceeded to the tagmentation step. We followed the standard protocol of 10X Multiome assay (10X Genomics) for  sample tagmentation and droplet generation. When performing the pre-amp PCR and cDNA amplification, 1μl of 100μM HTO additive primer (5' GTGACTGGAGTTCAGACGTGTGCTC-3') was added to help amplify the HTO. After cDNA amplification, save the supernatant from the 0.6X SPRI beads cleanup step to build the HTO libraries. The HTO library construction is following the standard cell hashing protocol.  For each 10X channel, 3 types of libraries were constructed: ATAC, GEX and HTO. The 3 types of libraries were sequenced on Illumina NovaSeq and NextSeq sequencers with roughly 30K, 20K and 2K raw reads per cell, respectively.