Nebnext rrna depletion kit human mouse rat

Cells were grown as above for 4sU-DRB sequencing without DRB treatment and with the duration of 200 μM 4sU treatment for 15 min. Total RNA extraction, biotinylation, streptavidin-based pull down and cleanup were done as described for 4sU-DRB sequencing.
cDNA libraries were prepared using Ultra Directional RNA Library Prep kits (New England Biolabs) and NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs) in 16-19 PCR cycles, depending on the 4sU-RNA input. PCR products were assessed for size distribution and concentration on a Bioanalyzer (Bio-Rad) or on the Fragment Analyzer (Advanced Analytical) using the NGS Fragment High Sensitivity Analysis Kit (1-6,000 bp; Advanced Analytical).

NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #Z1955E) was chosen to remove the targeted ribosomal RNA (rRNA). All RNA with a percentage of RNA fragments > 200 nucleotides (DV200) ≤ 50% skipped fragmentation and proceeded to library preparation. After rRNA depletion and fragmentation, cDNA synthesis and NGS library preparation were performed using NEBNext^® Ultra™ II Directional RNA Library Prep Kit (NEB#E7760L). The library was quantitated using Qubit 3.0 (life Invitrogen, USA) and quality was assessed with LabChip GX Touch (PerkinElmer, USA). After removal of terminal adaptor sequences and low-quality data by using fastp (version: 0.19.5) [15 (link)] and removal rRNA reads through aligning clean reads to rRNA database (download from NCBI) by using bowtie2 (version:2.2.8) [16 (link)], clean reads without known rRNA were aligned to the reference human genome (hg19) through STAR (version 020201) [17 (link)]. Fusions were detected by a customized version of Arriba 1.1.0. and annotated by in house software annoFilterArriba (version:1.0.0) with NCBI release 104 database. All final candidate fusions were manually verified with the integrative genomics viewer browser. A series of quality control metrics was computed by using RNA-SeQC assessment [18 (link)]. A threshold of ≥ 80 million mapped reads and ≥ 10 million junction reads per sample was set.

The H. pomatia snails were obtained from a farmers’ market in Germany and stored at -80° C. Approximately 100 mg of H. pomatia body were frozen in liquid nitrogen and ground to a powder. Total RNA was purified from rehydrated powder with the RNeasy Plant Mini Kit (Qiagen, Chatworth, CA). An mRNA transcript library for Illumina sequencing was created using the NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA). A NextSeq 500 sequencer (Illumina, San Diego, CA) was used to do paired-end deep sequencing (2X 150 bp) of the library. The utility CutAdapt [31 ] was used to remove adapter sequences from the raw reads. The trimmed reads were then assembled using the Trinity software package [32 (link)]. The assembled transcriptome data was deposited at DDBJ/EMBL/GenBank under the accession GKIM00000000. The version described in this paper is the first version, GKIM01000000. Raw reads were deposited in the NCBI Sequence Read Archive (accession #PRJNA936131).

Library preparation for RNA-Seq was performed using the NEBNext^® Ultra™ II Directional RNA Library Prep Kit for Illumina^® (New England Biolabs, Ipswich, MA, USA, E7760) starting from 36 ng of total RNA as input. Ribosomal depletion was achieved by using the NEBNext^® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA, USA, E6310) and indexing was performed by the use of NEBNext^® Multiplex Oligos for Illumina^® (New England Biolabs, Ipswich, MA, USA, E7335). Accurate quantification of the input RNA was performed with the QuantiFluor^® RNA System (Promega, Madison, WI, USA, E3310) and by TapeStation RNA ScreenTape Analysis (Agilent, Santa Clara, CA, USA, 5067-5576). Accurate quantification of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega, Madison, WI, USA). The size range of final cDNA libraries was determined applying the TapeStation D1000 ScreenTape (Agilent, Santa Clara, CA, USA, 5067-5582). cDNA libraries were amplified and sequenced using the NextSeq 500 with NextSeq 500/550 High Output Kit v2.5 (150 cycles; Illumina, San Diego, CA, USA, 20024907). Sequencing was performed as paired end; 2 × 76 bp; single indexing with ~30 million reads per sample.

Additional rRNA removal step was performed on n = 6 DNase treated RNA (two for each kit). The rRNA removal was performed using the NEBNext® rRNA Depletion Kit (Human/Mouse/Rat) (New England Biolabs, Ipswich, MA) as per the manufacturer’s instructions. Each viral extract was then subjected to Whole Transcriptome Amplification (WTA) using the QIAseq FX Single Cell RNA Library Kit (Qiagen, Hilden, Germany) as per manufacturer’s instructions to generate the cDNA. Amplified cDNA was quantified using the Qubit® DNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA) with the Qubit® 3.0 Fluorometer.

For library preparation, mRNA was isolated from DNAse-treated total RNA using the NEBNext rRNA depletion Kit (human, mouse, rat) from New England Biolabs (NEB) according to the manufacturer’s instructions. Final elution was done in 5 μl nuclease-free water. The samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (NEBNext Ultra II Directional RNA Library Prep; New England Biolabs). For ligation, custom adaptors were used (Adaptor-Oligo 1: 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3′, Adaptor-Oligo 2: 5′-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3′). After ligation, the adapters were depleted by XP bead purification (Beckman Coulter) adding beads in a ratio of 1:0.9. Dual indexing was done during the following PCR enrichment (15 cycles, 65°C) using custom amplification primers carrying the index sequence indicated with “NNNNNNN.” (Primer1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNNNN GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T). After two more XP bead purifications (1:0.9), libraries were quantified using the Fragment Analyzer (Agilent). Libraries were equimolarly pooled before sequencing them with a length of 75 bp in single end mode on an Illumina NextSeq 500 system to a depth of at least 40 mio reads.