Extended Data Fig. 10: Flow validation experiment of SCITO-seq.

a, To reduce the non-specific staining of secondary oligonucleotides, we titrated oligos at 1 μM (right) and 100 μM (left). After hybridization of oligo conjugated antibodies with a Cy5 conjugated reverse complementary oligo for 15 minutes, a mixture of LCLs and primary monocytes were stained with the hybridized material and CD13-BV421 for 30 minutes, washed twice and analyzed on a LSRII. CD13 BV421 antibody was captured by the Violet-F channel (x-axis) and Cy5 tagged secondary oligos was captured on the Red-C channel to check the level of background staining (Q6 gated population refers to the spillover of non-cognate secondary oligonucleotides in the primary monocyte population). b, To determine if 1 μl of 1 μM reverse complementary oligonucleotide would saturate 1 μg antibody, we first hybridized 1 μg of oligonucleotide conjugated CD3 with 1 μl of 1 μM reverse complementary oligonucleotide conjugated to Cy5. Following this, another 1 μl of 1 μM reverse complementary FAM conjugated oligo was added for 15 minutes prior to washing and analysis. The red population represents cells stained with sequentially hybridized antibody. The blue population represents cells stained with a conjugated CD3 antibody but without the hybridization sequence for the Cy5 or FAM oligo. c, Lymphocytes were gated for singlets and live cells (PI signal captured in the YG C-A channel) prior to binning samples across CD8a expression for sorting. Red-C represents CD8a-APC and Blue-B represents isotype control-AF488.