Next ultra 2 rna library prep kit

RNA was extracted from the CSF and blood B cell subsets using the AllPrep DNA/RNA Micro kit (Qiagen), automated on the QIAcube robotics platform. RNA-Seq libraries were prepared with the Next Ultra II RNA Library Prep kit (New England Biolabs), automated on a Beckman-Coulter Biomek NXP robot. Individual cDNA libraries were barcoded with dual-indexing barcodes, pooled evenly, and sequenced on an Illumina HiSeq 4000 machine to generate 150-base pair paired-end reads.

To investigate the possible causative pathogen or pathogens causing this disease, viruses isolated from an intestine and liver homogenate were blindly passed into 9-day-old embryonated goose eggs through allantoic cavity injection. After 10 passages, the allantoic fluid of infected eggs was collected and centrifuged at 8000 g/min for 20 min to remove cell debris. The supernatant was collected and further ultracentrifuged at 45,000 g/min for 2 h, and the pellet was resuspended with PBS and used for viral DNA/RNA extraction. Because the initial identification attempts yielded negative results, we performed a pathogen discovery protocol by using a next-generation sequencing instrument. The viral DNA and RNA were extracted separately using a QIAamp DNA extraction kit and QIAN Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. Concentrations of the extracted RNA were quantified by using a Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA). The extracted samples were used to prepare the libraries by using NEB Next Ultra II RNA Library Prep Kit (NEB, Beijing, China). Similarly, we prepared paired-end DNA sequencing libraries from the DNA samples using a Nextera XT DNA Library Prep Kit (Illumina).