Nebnext rrna depletion kit v2

For RNA-seq from mESCs, total RNA was extracted from one well of a six-well plate of 60%–80% confluent cells with QIAzol reagent following the manufacturer's recommendation. Libraries were prepared with NEBNext Ultra II Directional RNA Library Kit for Illumina with prior use of NEBNext rRNA Depletion Kit v2 (E7405, NEB) following the manufacturer's protocol. Libraries were amplified using eight PCR cycles and sequenced on a NextSeq 500 (Illumina) in 75 bp single-end read mode.
For downstream analyses, adaptor sequences were removed from the reads with cutadapt (Kechin et al. 2017 (link)) (1.18) using default settings. For the analysis of differentially expressed retrotransposons, consensus sequences of rodent retrotransposons were retrieved from Repbase (24.01) and used to map the processed reads using bowtie2 (Langmead and Salzberg 2012 (link)) (2.4.2) with default settings. The number of mapped reads per retrotransposon were counted and analyzed using DESeq2 (1.32.0) (Love et al. 2014 (link)).

Total RNA was extracted from lysates using Direct-zol RNA Miniprep Kit (Zymo) and NEBNext Ultra II Directional RNA Illumina Library Prep Kit (E7760; NEB) with NEBNext rRNA Depletion Kit V2 (E7400; NEB). RNA-seq samples were prepared with single index primers (ME6609S; NEB). RNA integrity and concentration of amplified libraries were assessed with a Bioanalyzer and Qubit; 5 ng/sample was pooled and sequenced with the Illumina HiSeq 4000 (SE65 66x8x8x0) via the sequencing core of the UCSF Center for Advanced Technology.

RNA was extracted from cells using TRI Reagent (Molecular Research Center, Inc) and further purified using Zymo RNA clean and concentrator-5 kit (Zymo Research) with DNase I treatment. The ribosomal RNAs were removed using NEBNext rRNA Depletion Kit v2 (New England Biolabs) before subjecting the samples to sequencing library construction. Strand-specific RNA sequencing libraries were prepared as described previously (Borodina et al, 2011 (link)).

RNA was prepared as previously described^{27 (link)}. In summary, small sections of cotton rat lung (lingular lobe), large intestines (~ 20 mm colon, flushed with sterile PBS), spleen, kidney, and heart were homogenized using a NextAdvance Bullet Blender® with RED RINO™ tubes containing 3.2 mm stainless steel beads (SSB32) and 2 × volume of QIAzol® Lysis Regent (Qiagen, cat. no. 79306) at max speed for 3 min. RNA was extracted from homogenates using RNeasy® Plus Universal Mini kit (Qiagen, cat. No. 73404) via additional QIAzol® (total volume 900uL), gDNA eliminator, and chloroform with column DNase digestion as recommend by the manufacturer’s protocol. RNA quality was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Host rRNA was removed using the NEBNext rRNA Depletion Kit v2 (Cat no: NEB E7400X). The cDNA libraries were prepared using 1 μg total RNA from each sample using the NEBNext Ultra II RNA Library Prep Kit for Illumina (Cat no: NEB E7805L) following the manufacturers protocol for intact RNA (RIN > 7), AMPure XP Beads for cleanup steps (Beckman, cat. No. A63881), and NEBNext Multiplex Oligos for Illumina (Set 1, cat no: E7600S) for pooling samples. Sequencing was performed using Illumina NovaSeq6000 2 × 150 bp sequencing at the Vanderbilt Technologies for Applied Genomics (VANTAGE) core facility.

The extracted RNA of three RT-PCR positive bovine serum samples (BovHepV_Bulgaria 9/19/313) and the nested RT-PCR positive red deer serum sample was subjected to next generation sequencing (NGS) analysis. RNA Illumina NGS libraries were prepared from each sample after rRNA removal using the NEBNext rRNA Depletion Kit v2 followed by NEB Ultra II RNA library preparation (New England Biolabs) according to manufacturer’s instructions. Libraries were multiplex-sequenced on an Illumina MiSeq instrument (300 cycles, PE protocol) with approximately 4,000,000 reads per sample. Bioinformatic analysis of the obtained short read files was performed as previously published using our inhouse Pathogen Detection Tool DAMIAN [40 (link)].

Total RNA was isolated from 10×10 μm thin slides of paraffine-embedded lungs stocked at room temperature. NucleoSpin total RNA FFPE XS Micro kit (Cat. 740969.50, Macherey-Nagel) was used according to manufacturer’s instruction, and RNA was eluted in 30 μL final volume. RNA concentration was assessed after extraction at the NanoPhotometer® N60 (Implen). RNA integrity (>50% DV200) was assessed by Bioanalyzer (total RNA Pico Kit and RNA 6000 Nano Kit, 2100 Instrument, Agilent Technologies, Paolo Alto, CA, USA). Sequencing libraries were prepared using “NEBNext Ultra II Directional RNA Library Prep Kit for Illumina “combined with “NEBNext rRNA Depletion Kit v2” for ribosomal RNA depletion (New England Biolabs, Ipswich, MA). Libraries were pooled and sequenced (paired-end, 2 × 100bp) on a NextSeq2000 according to the manufacturer’s instructions (Illumina Inc., San Diego, CA, USA).