Nebnext ultra 2 non directional rna second strand module

RNA was extracted from bat fecal samples using the QIAamp Viral RNA Mini Kit (Qiagen, Germany). RNA carrier was dissolved in AVE buffer and added to AVL buffer according to manufacturer’s recommendations before extraction. 140 μL of fecal sample was added to the prepared AVL buffer with carrier RNA–Buffer AVE. Further steps were performed according to the original protocol. RNA was eluted with 60 μL of the AVE buffer and stored at −70 °C until evaluation. 10 μL of RNA was used for reverse transcription using Reverta-L reagents (AmpliSens, Russia). Second strand cDNA was obtained using the NEBNext Ultra II Non-Directional RNA Second Strand Module (E6111L, New England Biolabs). In order to increase input concentration, 24 μL of first strand product was added to 10 μL of H2O (milliQ) for subsequent steps.

RNA extracts were converted to cDNA using Protoscript II First Strand cDNA Synthesis Kit (New England Biolabs Inc.), NEBNext Ultra II Non-Directional RNA Second Strand Module (New England Biolabs Inc.), and Random Primer 6 (New England Biolabs Inc.). A total of 25 ng of the cDNA and DNA mix was used for library construction using Twist Library Preparation EF 2.0 Kit and Twist Universal Adaptor System (Twist Biosciences). The libraries were pooled, a maximum of 16 samples per pool, at equal mass to a total 1,500 ng per pool. The Twist Comprehensive Viral Research Panel (Twist Biosciences) was used to hybridize the probes at 70 °C for 16 h. The post-capture pool was further PCR amplified for 12 cycles and final libraries were sequenced on Illumina NovaSeq 6000 SP flow cell, to generate 2×150 bp paired-end reads. Following sequencing, raw data files in binary base call (BCL) format were converted into FASTQs and demultiplexed based on the dual-index barcodes using the Illumina ‘bcl2fastq’ software.