Single-cell suspension was loaded on a Chromium Controller Instrument (10x Genomics) to generate single-cell gel bead-in-emulsions (GEMs). Barcoding and complementary DNA (cDNA) libraries were prepared using the Single-Cell 3′ Library & Gel Bead Kit, according to the manufacturer’s instructions. GEM-reverse transcription (RT) was performed in a Mastercycler Nexus Thermal cycler (Eppendorf). After RT, GEMs were broken, and the cDNA was cleaned up with Dynabeads MyOne Silane Beads (Thermo Fisher Scientific) and amplified. Amplified cDNA product was cleaned up with the SPRIselect Reagent Kit (0.6× SPRI; Beckman Coulter). Indexed sequencing libraries were constructed using the Chromium Single-Cell 3′ Library Kit (10x Genomics) for enzymatic fragmentation, end-repair, A-tailing, adaptor ligation, post-ligation cleanup, sample index polymerase chain reaction (PCR), and PCR cleanup. Final libraries were quantified by Qubit, TapeStation, and quantitative PCR using NEBNext Library Quant Kit (BioLabs). Libraries were loaded at 2 to 2.2 pM on an Illumina NextSeq500 using the high-output 75-cycle kit with the following parameters: 26 bp for Read1, 56 bp for Read2, and 8 bp for I7 Index. Each library was sequenced twice using Illumina’s NextSeq 500 sequencing platform. Sequencing depth was 50,000 to 100,000 reads per cell.

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