Total RNA samples were treated with DNAse I using Turbo DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the kit guidelines. Libraries for RNA sequencing were prepared using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with the NEBNext rRNA Depletion Module (New England Biolabs, Ipswich, MA, USA). RNA libraries were sequenced on the HiSeq system (Illumina, San Diego, CA, USA) with 250 cycles. Sequenced reads were pseudoaligned to hg38 gencode (v37) transcriptome using kallisto v0.48.0 [42 (link)] with default settings. Gene level expression abundances were calculated using the tximport Bioconductor package [43 (link)]. Expression of the CDKN1A gene was presented in transcripts per kilobase million (TPM).
Nebnext rrna depletion module
Manufactured by New England Biolabs
Sourced in United States
The NEBNext rRNA Depletion Module is a laboratory tool designed to selectively remove ribosomal RNA (rRNA) from total RNA samples. This module can be used to enrich for non-rRNA species, such as messenger RNA (mRNA) and non-coding RNAs, prior to downstream applications like RNA-sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Turbo dna free kit, by Thermo Fisher Scientific (4 mentions)
Nebnext ultra 2 directional rna library prep kit, by Illumina (2 mentions)
Hiseq system, by Illumina (2 mentions)
Agilent 2100 bioanalyzer rna nano chip, by Agilent Technologies (1 mentions)
Nebnext ultra 2 directional rna kit, by New England Biolabs (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
3 protocols using nebnext rrna depletion module
1
RNA-seq Protocol for CDKN1A Expression
2
Comprehensive RNA-seq Library Preparation
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Total RNA was prepared with RNeasy Mini kit (QIAGEN) and quantified using the Qubit 2.0 Fluorometer (Invitrogen) and evaluated on the Agilent 2100 Bioanalyzer RNA nano chip (Agilent Technologies). All RNA samples were excellent quality with RNA Integrity Number >8.0. The DNase-treated Total RNA (300 ng input) was depleted of ribosomal RNA using probes provided by the NEBNext rRNA depletion module (#E6310L; NEB) according to manufacturer recommendations. The library preparation from the rRNA-depleted RNA was conducted according to NEBNextUltra II Directional RNA kit (#E7760; NEB) guidelines. The final libraries were validated on the bioanalyzer DNA chips, normalized to 2 nM, pooled equally and loaded onto the NextSeq 500 v2.5 flow cell at 1.8 pM final concentration and sequenced using 2 × 80 bp paired-end chemistry. On average, 20–25 million reads pass filter (base quality score >Q30 suggesting less than one error in 1,000 bp) are generated per sample. The RNA-seq data was deposited in the Gene Expression Omnibus under accession no. GSE208321 .
3
RNA-seq Library Preparation and Sequencing
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Total RNA samples were treated with the DNAse I using Turbo DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the kit guidelines. Libraries for RNA sequencing (RNA-seq) were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina with the NEBNext rRNA Depletion Module (New England Biolabs, Ipswich, MA, USA). RNA libraries were sequenced on the HiSeq system (Illumina, San Diego, CA, USA) in a paired-end mode with the read length of 125.
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