Blood draws were performed in the morning under fasting conditions. Approximately 2.5 mL of whole blood were kept frozen at −80oC in a PAXgene tube (QIAGEN, 761115) until processing (Jefferson et al., 2016 (link)). RNA extraction, library preparation, and RNA sequencing were performed by the VANTAGE Core (Vanderbilt University, TN, USA). Total RNA was extracted from whole blood using the QIASymphony RNA Kit (QIAGEN, 931636), and both ribosomal RNA and hemoglobin were depleted with the NEBNext Globin and rRNA Depletion Kit (New England BioLabs, Inc., E7750). Library preparation was completed using the NEBNext Ultra Directional Library Prep Kit (New England BioLabs, Inc., E7420) before sequencing was performed using 150 base pair (bp) paired end reads on an Illumina NovaSeq 6000 (Illumina), targeting an average of 50 million reads per sample.
Nebnext globin and rrna depletion kit
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Globin and rRNA Depletion Kit is a laboratory tool designed to selectively remove globin and ribosomal RNA (rRNA) sequences from RNA samples. This kit helps improve the quality and diversity of RNA sequencing data by reducing the over-representation of these abundant transcripts.
Automatically generated - may contain errors
Lab products found in correlation
Qiasymphony rna kit, by Qiagen (2 mentions)
Paxgene tube, by Qiagen (2 mentions)
Nebnext ultra directional library prep kit, by New England Biolabs (2 mentions)
Tri reagent ls, by Merck Group (2 mentions)
Hiseq 1500, by Illumina (2 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
4 protocols using nebnext globin and rrna depletion kit
1
RNA-seq of Whole Blood Transcriptome
2
Transcriptome Profiling of Keds
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Individual specimens of M. ovinus were homogenized using Tissue Lyser 2 (12 min, frequency 25 s−1). Prior to extraction of the nucleic acid, aliquots of the homogenate of keds collected from the same animal were pooled together in equal amounts. RNA was extracted from pooled homogenates using TRI Reagent LS (Sigma, St. Louis, MA, USA) according to the manufacturer’s instructions. After extraction, host rRNA was depleted using a NEBNext Globin and rRNA Depletion Kit (NEB, E7750S, Ipswich, MA, USA) according to the manufacturer’s instructions. The obtained RNA was used for library preparation without polyA-enrichment using a NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770, Ipswich, MA, USA) according to the manufacturer’s instructions. Final libraries were sequenced (single-end, 250-nt reads) on a HiSeq1500 (Illumina, San Diego, CA, USA). Raw reads were deposited in the sequence read archive (BioProject accession number PRJNA777535).
3
Termite Virome Extraction and Sequencing
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Termite specimens were homogenized in the pools (Table 1 ) using Tissue Lyser 2 (Qiagen, Hilden, Germany) for 15 min with frequency 25 s−1. High-throughput sequencing and the data-processing pipeline were the same as described previously [15 (link)].
RNA was extracted using TRI Reagent LS (Sigma, St. Louis, MA, USA) according to the manufacturer’s manual. After the extraction, host rRNA was depleted using an NEBNext Globin and rRNA Depletion Kit (NEB, E7750S, Ipswich, MA, USA) according to the manufacturer’s instructions. The obtained RNA was used for library preparation without polyA-enrichment using a NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770, Ipswich, MA, USA) according to the manufacturer’s instructions. Final libraries were sequenced (single-end, 250-nt reads) on a HiSeq1500 (Illumina, San Diego, CA, USA), and raw reads were deposited under the BioProject accession number PRJNA817887.
RNA was extracted using TRI Reagent LS (Sigma, St. Louis, MA, USA) according to the manufacturer’s manual. After the extraction, host rRNA was depleted using an NEBNext Globin and rRNA Depletion Kit (NEB, E7750S, Ipswich, MA, USA) according to the manufacturer’s instructions. The obtained RNA was used for library preparation without polyA-enrichment using a NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, E7770, Ipswich, MA, USA) according to the manufacturer’s instructions. Final libraries were sequenced (single-end, 250-nt reads) on a HiSeq1500 (Illumina, San Diego, CA, USA), and raw reads were deposited under the BioProject accession number PRJNA817887.
4
RNA-Seq Analysis of Whole Blood Samples
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