Viral Sequence Capture Library Prep
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Corresponding Organization :
Other organizations : Columbia University
Protocol cited in 12 other protocols
Variable analysis
- Viral sequence capture
- Reverse transcription using SuperScript III with random hexamers
- RNase H treatment prior to second-strand synthesis with Klenow fragment
- Shearing of the cDNA/DNA mix to an average fragment size of 200 bp using Covaris E210 focused ultrasonicator
- Library preparation using KAPA library preparation kits with Roche/NimbleGen adapter kits
- Hybridization with SeqCap HE universal oligonucleotide, SeqCap HE index oligonucleotides, and COT DNA
- Denaturation at 95°C for 10 min
- Hybridization with VirCap probe library at 47°C for 12 h
- Capture with SeqCap Pure capture beads at 47°C for 45 min
- Washing of the capture beads with buffers of increasing stringency
- Post-hybridization PCR with SeqCap EZ accessory kit V2
- Quality and quantity of libraries checked using a Bioanalyzer (Agilent)
- Illumina sequencing of the purified PCR products
- Standard KAPA protocol
- SeqCap RNA enrichment system protocol (Roche/NimbleGen)
- Positive control: None specified
- Negative control: None specified
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