title: GSM7025095: scProgenyProduction_highMOI_polyAseq; Canis lupus familiaris; Influenza A virus (A/California/04/2009(H1N1)); RNA-Seq
library_construction_protocol: All code for data processing as well as detailed explanations are at https://github.com/jbloomlab/barcoded_flu_pdmH1N1. Single-cell RNA sequencing was performed with the 10X Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1. The manufacturer's standard protocol was used with the following modifications. The template-switching oligo was replaced with a modified single-stranded DNA oligo with the sequence 5'-AGAGTGTTTGGGTAGAGCAGCGTGTTGGCATGTrGrGrG-3' at a final concentration of 45 uM in the reaction mix. This change was made to accommodate some of the barcoded influenza segments' exogenous sequence which shares homology with the standard 10X template-switching oligo. The cDNA amplification primer mix was replaced with a pair of primers with the sequences 5'-AGAGTGTTTGGGTAGAGCAGCG-3' (binding to the custom template-switch oligo mentioned above) and 5'-CTACACGACGCTCTTCCGATCT-3' (binding to the standard 10X adapter sequence) at a final concentration of 1 uM in the reaction mix. The cDNA amplification PCR reaction extension time was increased to 20 seconds to encourage the formation of full-length cDNA products. The amplified cDNA product was split in half; one half was used for fragmentation and preparation of the transcriptome sequencing library while the other half was used as template for long-read sequencing of viral transcripts.  At 12 hours post-infection, the supernatant was collected and cells and debris were removed by centrifugation at 300 RCF for 3 min. The supernatant was split into 2 aliquots of 220 ul each and frozen at -80C. The cells were collected by addition of 100 ul trypsin and a single-cell suspension was generated. The trypsin digestion was stopped by addition of 400 ul of D10 medium. The cells were washed 3 times with phosphate-buffered saline supplemented with 0.8% by volume non-acetylated bovine serum albumin. The cells were counted to confirm that approximately 7,000 cells were present per well and loaded into the 10X Chromium device.