Hiseq rapid run mode

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)

Genomic DNA of the cells were purified using Qiagen DNeasy Blood and Tissue Kits. To amplify the dual gRNAs from each sample, we used 20 μg of genomic DNA as template across ten 50-μL PCR reactions with Kapa Hifi polymerase. By testing the amplification efficiency, we used 22 – 24 cycles at an annealing temperature of 55 °C with the following primers:
The amplicons were pooled and purified with Agencourt AMPure XP bead at a double selection of 0.55× and then 0.8×. The samples were quantified with Qubit dsDNA High Sensitivity Kit. To attach Illumina sequencing adaptors and indexes, we used 50 ng of purified step I PCR product as template across four 50-μL PCR reactions with Kapa Hifi polymerase using primers of Next Multiplex Oligos for Illumina (New England Biosciences). 7 or 8 PCR cycles were carried out at an annealing temperature of 72 °C. The PCR product were purified twice with Agencourt AMPure XP bead at 0.8× ratio, quantified, pooled and sequenced on an Illumina HiSeq rapid-run mode for 75 cycles paired-end runs.