Bulk DNA-seq of Homo sapiens; Viral lineage marker abundance estimation library, APC-/-,TP53-/-,KRAS G12D,SMAD4-/-, passage 16
For each organoid line DNA was harvested weekly. Cell lysis was performed overnight at 50C using 0.05 units of Qiagen Protease in 10 mM tris pH 7 in a total volume of 1 ml. All samples were split into two and DNA was extracted using phenol/chloroform extraction followed by AMPure DNA bead clean-up (Beckman). Viral barcodes were amplified using a two-step PCR strategy (Levy et al., 2015). All PCRs were done in 96 well plate, using the 96 barcoded forward primers (1 primer per well, supplementary table 2) in combination with a mix of 5 reverse primers. The 5 reverse primers are identical except for a small (No insert, 1, 2, 3 or 4 base insertion), which ensures high complexity of the libraries, required for sequencing. First, for both replicates of a sample 5 cycles of PCR were performed on 500ng of genomic DNA using NEBnext High Fidelity PCR master mix (NEB) and barcoded primers containing a Unique Molecule Identifier (UMI) (Supplementary table 2). After PCR, excess primers were digested using ExoSap (Agilent) to prevent UMI replacement during later stages of amplification. After ExoSap treatment PCR reactions were cleaned up using AMPure beads and another 25 cycle PCR was performed using Illumina Truseq small RNA library PCR primers. Libraries were sequenced on Illumina NextSeq 500 using 2x75 bp paired end sequencing.. Viral barcode in read 1, read 2 contains 8bp UMI and 8bp library identifier