title: GSM7025099: scProgenyProduction_highMOI_supernatant_NA_biolRep2; Influenza A virus (A/California/04/2009(H1N1)); OTHER
library_construction_protocol: All code for data processing as well as detailed explanations are at https://github.com/jbloomlab/barcoded_flu_pdmH1N1. RNA was isolated from the viral supernatant or infected cell pellets using the RNeasy Mini Kit. Lysis buffer was mixed with the viral supernatant sample and 70% ethanol was added. For the infected cell pellets, the sample was mixed with lysis buffer and homogenized by vortexing at high speed for 20 seconds. The homogenized sample was processed on a gDNA eliminator spin column to remove genomic DNA. The processed sample was combined with 70% ethanol. From this point, both the viral supernatant and infected cell pellets were treated identically and followed the standard RNA purification protocol specified by the manufacturer. The RNA for each sample was eluted in 50 ul of RNase-free water. Reverse transcription was performed with a segment-specific primer targeted to the HA or NA vRNA. Two replicate reactions were performed using RNA from the viral supernatant sample, and two independent reactions were performed using RNA from the two infected cell pellet samples; these replicates provide technical duplicate measurements of both the physical progeny in the supernatant and the infectious progeny in the cell pellets. Reverse transcription was performed using the SuperScript III First-Strand Synthesis SuperMix kit according to the manufacturer protocol [46]. For the viral supernatant samples, 12 ul of each RNA sample was used as template for each 40 ul reaction. For the infected cell pellet samples–which contain much larger amounts of total RNA, due to the host RNA present in the cell–1000 ng of RNA was used as template for each 40 ul reaction. The low-concentration cDNA generated from the viral supernatant samples was purified and concentrated using 2X Ampure SPRI beads and eluted into 22 ul of elution buffer. Viral barcodes were amplified in 50 ul PCR reactions using KOD Hot-Start Master Mix. For the viral supernatant samples, 22 ul of concentrated cDNA was used as template. For the high-concentration infected cell pellet samples, 10 ul of unpurified cDNA was used as template. Segment-specific primers (Table S3) were used and reactions were run for 20 cycles. Amplicons were size-selected and purified using a double-sided AmpureXP bead cleanup. Samples were first combined with 0.8X AmpureXP beads and the supernatant was collected. The supernatant was then combined with 1.8X AmpureXP beads and the bound DNA was collected. Sequencing indices and adapters were attached in a 50 ul PCR reaction using KOD Hot-Start Master Mix. For all samples, 2 ng of purified amplicon DNA was used as template. Sample-specific index primers (Table S3) were used and reactions were run for 20 cycles. The resulting amplicons were gel-purified and pooled for sequencing. Single-end sequencing was performed on an Illumina MiSeq.  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.