Nebnext library preparation kit

Frozen tissue from nine resected PDACs (four diploid and five polyploid) that were biallelically mutant for TP53 were processed for single-nucleus isolation as previously described^{56 (link)}. Research involving resected samples was approved by the MSKCC institutional review board (MSKCC IRB). In brief, two 50 μm tissue sections were cut and mechanically dissociated using a pair of scalpels in the presence of 1 ml of nuclei isolation buffer (NST-DAPI). DAPI-stained nuclei were subsequently sorted on the basis of DNA content with single-nuclei collected from the diploid and polyploid gate (when present) and deposited into 96-well plates preloaded with nucleus lysis buffer as described above. Single-nucleus WGA amplification was performed as described above for mouse SP and DP single cells with downstream sequence library performed using the NEBNext Library preparation kit (NEB). Libraries were sequenced on the HiSeq2500 instrument for single-end 101 bp sequencing while targeting a coverage of around 1–2 million reads per single cell. Single-cell demultiplexed sequencing data were processed for absolute copy-number determination as previously described^{58 (link)}. The absolute copy number was converted to the nearest integer, and homogeneity score was inferred as the frequency of the major integer copy-number state for each bin across the genome.

Genomic DNA was prepared using AllPrep DNA/RNA mini kit (QIAGEN), fragmented by sonication (Covaris) and ligated to methylated adapters (Illumina) with the NEBnext library preparation kit (New England BioLabs). DNA was subsequently bisulfite-treated using the Sigma Imprint kit according to the manufacturer’s instructions (one step protocol). Final library amplification (11 cycles) was performed with KAPA Uracil+ (Kapa Biosystems), after which the libraries were purified using 1x Ampure beads. Sequencing reads were filtered to remove low-quality calls and adapters were removed using v0.2.2 of Trim Galore (www.bioinformatics.babraham.ac.uk/projects/trim_galore) with default parameters. The remaining sequences were mapped to the human reference genome GRCh37 using Bismark v0.7.4 (Krueger and Andrews, 2011 ) with default parameters, and CpG methylation calls were extracted and analyzed using SeqMonk (www.bioinformatics.babraham.ac.uk/projects/seqmonk) and custom R scripts. Global methylation comparison was calculated by averaging 1kb window methylation levels of CpGs covered by at least 30 reads. To determine CGI methylation percentages probes were generated over CGIs and filtered for a minimum of 1 methylation count/CG and at least 5 CGs/CGI. Methylation values represent the mean over each CGI, filtered by chromosome.

Viro-1 and Viro-2 mutants were crossed with the WM27 rde-1(ne219) strain, and F1 progenies were chosen and transferred to six-well plates. After 3 days, the F2 progenies were challenged with Orsay virus. A total of 72 F2 Viro animals were picked off F1 plates and transferred to six-well plates. F2 plates were replicated after 1 day of laying eggs. The original F2 plates were challenged again with Orsay virus, and both virus-induced GFP fluorescence and Orsay virus RNA levels were assayed at 3 days postinfection. Wells that yielded exclusively Viro animals and that yielded low RNA levels from replicated F2 plates were pooled, and genomic DNA from pooled samples was extracted using a Qiagen genomic DNA preparation kit according to the manufacturer’s protocol. DNA libraries were prepared using NEB-Next library preparation kit (New England Biolabs) and then sequenced using Illumina Hiseq 2500. The raw sequence data were analyzed using the pipeline CloudMap for C. elegans gene mapping (16 (link)).