RNA samples were tested for quality on the Agilent Tapestation 2200 (Agilent Technologies, Santa Clara, CA). The NEB Next rRNA Depletion Kit (NEB #E6310X) was used to process 250 ng of total RNA. RNA-Seq libraries (400 bp) were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760L). Samples were individually indexed using the NEBNext Multiplex Oligos for Illumina (NEB #E6609S). Adapter ligated DNA was amplified in 13 cycles of PCR enrichment. Libraries were quantified with the Quanti-iT dsDNA HS Kit (Invitrogen) and qPCR. Library validation was performed on the Agilent 2200 Tapestation. Independently indexed stranded cDNA libraries were pooled and sequenced for 150 cycles with the Illumina NovaSeq 6000 S2 Kit. All samples were sequenced at 85–90 million read depth, paired-end 2 × 150 bp, and in reverse-stranded orientation.
Next rrna depletion kit
Manufactured by New England Biolabs
The Next rRNA Depletion Kit is a laboratory product designed to selectively remove ribosomal RNA (rRNA) molecules from RNA samples. The kit utilizes a hybridization-based approach to deplete rRNA, allowing for the enrichment of other RNA species, such as messenger RNA, for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2200 tapestation, by Agilent Technologies (1 mentions)
Multiplex oligos, by New England Biolabs (1 mentions)
Nebnext ultra 2 directional kit, by New England Biolabs (1 mentions)
Nebnext ultra directional rna library prep kit, by Illumina (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Novaseq 6000 system, by Illumina (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Xp bead purification, by Beckman Coulter (1 mentions)
4 protocols using next rrna depletion kit
1
RNA-Seq Library Preparation and Sequencing
2
RNA Purification and Sequencing Preparation
3
RNA-seq Analysis of B Cell Activation
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CD19+ B cells were stimulated with CpG (1 μM) and IL-2 (25 U ml−1) ± GGTi-298 for 12 h. RNA was isolated by column centrifugation using RNeasy Plus mini kit (with gDNA removal step) as per manufacturer’s instructions (Qiagen). Library preparation was completed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina. Depletion of ribosomal RNA was performed using Next rRNA Depletion kit (New England BioLabs). RNA quality was confirmed by bioanalyser (Agilent 2100 Bioanalyzer G2938B), resulting in a mean RIN score of 8.1, ranging from 7.3 to 8.7. Paired-end sequencing was then conducted using the HiSeq 2500 platform (Illumina). Raw data were checked for quality using FASTQC. Processing of the raw data involving alignment (STAR) and annotation (hg19) were done using Partek. After annotation, TMM-normalized data were used for downstream statistical analysis using the exact test in the edgeR package. Inclusion criteria for significantly differentially expressed genes was a false discovery rate of <0.05 and a fold change of greater than 1.5×. Subsequent processing and visualization of the data was completed in RStudio or Morpheus (Broad Institute, Boston, MA).
4
Directional RNA-Seq Library Prep
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Only total RNA with RNA-integrity numbers ≥ 9.5 was used. mRNA was isolated from 370 ng DNAse treated total RNA using the Next rRNA depletion Kit (New England Biolabs) according to the manufacturer’s instructions. Samples were subjected to the workflow for strand specific RNA-Seq library preparation (Next 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 adapters were depleted by an XP bead purification (Beckman Coulter) by adding bead in a ratio of 1:0.9. Unique dual indexing was done during the following PCR enrichment (12 cycles) using custom amplification primers carrying the index sequence indicated with ‘NNNNNNN’ (Primer 1: AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, Primer 2: 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 beads purifications (1:0.9), libraries were quantified using the Fragment Analyzer (Agilent). Libraries were equimolarly pooled before sequencing them on an Illumina NovaSeq 6000 system in 100 bp paired-end mode to a depth of at least 40 million fragments.
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