Nebnext ultra 2 fs dna kit

For viruses, RNA extraction and amplification of nearly full-length genomes using 5 overlapping RT-PCRs were performed as described previously [22 (link)]. The DNA from the five PCR reactions was purified using the Zymo DNA clean&concentrator-25 kit and pooled in equal amounts.
Library preps for Illumina sequencing were prepared using the NEBnext ultra II FS DNA kit (New England Biolabs, Ipswich, MA, USA). For both PCR pools and plasmids, 25 ng of DNA were used as input. Library preps produced fragments of 500–600 bp and were sequenced on a Miseq using the 250 bp pair-end setting with the v2 500 cycles sequencing kits (Illumina, San Diego, CA, United States). Sequence analysis was performed as described previously for rescued viruses [26 (link)] but using Cutadapt [27 (link)] to trim off internal primer sequences located at the 5′-end of the read. Plasmid construct sequences were confirmed in the Geneious Prime software (Auckland, New Zealand).

The RNA of 1,000-3,000 freshly sorted cells was extracted using Arcturus PicoPure kit (Applied Biosystems) and DNase treatment was performed (Qiagen). Subsequently, cDNA libraries were prepared using SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) with 12 cycles of amplification. Further, NEBNext Ultra II FS DNA kit and NEBNext Multiplex Oligos (New England BioLabs) were used to generate uniquely dual-barcoded sequencing libraries from cDNA libraries. To this end, 5 ng of cDNA library was fragmented for 22.5 min, adaptors were ligated and libraries were amplified for eight cycles. RNA-seq libraries were sequenced at a depth of 55 million reads, 100 bp paired end on a NovaSeq platform (Illumina).