scRNA-seq for Donor 3, hashed perturbation sample 12
Briefly, a total of 12,000 cells (1,200 per multiplexing oligo) were loaded into a single channel of a Chromium Next GEM Chip G (10X Genomics, Cat \# 1000120) and partitioned into droplets with gel beads using a Chromium controller (10X Genomics, cat \# 1000204).  After emulsion droplets were formed and collected, reverse transcription reactions were incubated at 53C for 45 minutes.  Barcoded transcripts were purified, amplified and size fractionated to create separate libraries for the transcriptome and feature barcode fractions from each sample.  Transcriptome libraries were fragmented and ligated to indexed sequencing adapters according to the manufacturers recommended protocol.  Feature barcode libraries were prepared using 3 Feature Barcode Kit (10X Genomics, cat \# 1000262) following the manufacturers recommended protocol (10X Genomics, CG000388 Rev C).  Transcriptome libraries were sequenced on an Illumina NovaSeq6000 with paired end reads as follows:  Read 1 = 28 cycles, i7 Index = 10 cycles, i5 = 10 cycles, Read 2 = 89 cycles.  Feature barcode libraries were sequenced on an Illumina NovaSeq6000 with paired end reads as follows:  Read 1 = 28 cycles, i7 Index = 10 cycles, i5 = 10 cycles, Read 2 = 35 cycles.  Cell Ranger (v5.0.1) mkfastq was used to generate all demultiplexed FASTQ files from the raw sequencing data.