For each NextSeq sequencing run, raw sequencing data were converted to FASTQ files using bcl2fastq2 software (Illumina). Each sequencing sample was demultiplexed using Nextera N7xx indices. Tagging, trimming, alignment, and adding annotation tags were performed according to the standard Drop-Seq pipeline (http://mccarrolllab.org/dropseq/). Briefly, reads were first tagged according to the 12-bp cell barcode sequence and the 8-bp unique molecular identifier (UMI) in “read 1.” Then, reads in “read 2” were aligned with the hg19 or hg19-mm10 concatenated reference depending on the experiments and collapsed onto 12-bp cell barcodes that corresponded to individual beads. A Hamming distance of 1 was used to collapse UMI within each transcript. Digital expression matrix was obtained by collapsing filtered and mapped reads for each gene by UMI sequence within each cell barcode.

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