CITE-Seq cDNA Library Construction

cDNA library construction following GEM formation was performed as directed by 10X using v2/3 3’ chemistry depending on experiment. Following the published CITE-Seq protocol, an additive primer (partial Read 2 small RNA) was spiked into the cDNA amplification reaction. During the post-cDNA amplification SPRI cleanup step, given that Zipcode reads are significantly shorter (<200 bp), these reads were separated from cDNA reads by decanting the supernatant. cDNA reads bound to SPRI beads were processed as recommended in the 10X use guide. Meanwhile the supernatant containing Zipcode reads was saved and underwent two successive 3X SPRI cleanup steps.. This library was then amplified using primers from CITE-Seq protocol^{8 (link)}. Following fragment analysis on the BioAnalyzer and library quantification by qPCR, the Zipcode library was mixed with the associated cDNA library at a 1:10 molar ratio and sequenced on either Illumina HiSeq Rapid Run mode (all studies save for Fig. 4) or NovaSeq SP (Fig.4 studies) using 10X recommended sequencing parameters based on kit version (v2 vs. v3)