CD4+ T cells were isolated using negative selection (Stem Cell Technologies CD4 enrichment kit) according to the manufacturer’s recommendations. Total RNA was extracted using the Qiagen RNA Kit with Qiashredder columns. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size and integrity analysed on the 2200 TapeStation (Agilent, RNA ScreenTape). Input material was normalised to 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA Kit (NEB) following manufacturer’s instructions. Libraries were amplified on a Tetrad (Bio-Rad) using in-house unique dual indexing primers43 (link). Individual libraries were normalised using Qubit, and the size profile analysed on the 2200 TapeStation. Sequencing was performed using an Illumina Novaseq6000 platform at 150 paired end mode (Illumina, San Diego, CA).
Nebnext ultra 2 mrna kit
Manufactured by New England Biolabs
The NEBNext Ultra II mRNA kit is a laboratory product designed for the preparation and enrichment of messenger RNA (mRNA) from various sample types. The kit provides a streamlined workflow for the conversion of RNA into a sequencing-ready library.
Automatically generated - may contain errors
Lab products found in correlation
Fluostar optima plate reader, by BMG Labtech (3 mentions)
Ribogreen, by Thermo Fisher Scientific (3 mentions)
Tetrad, by Bio-Rad (3 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Rneasy fibrous tissue mini kit, by Qiagen (1 mentions)
Hiseq 3000 4000 pe cluster kit, by Illumina (1 mentions)
150 cycle sbs kit, by Illumina (1 mentions)
Rna screentape, by Agilent Technologies (1 mentions)
3 protocols using nebnext ultra 2 mrna kit
1
RNA-Seq Protocol for CD4+ T Cells
2
RNA-seq from Frozen Heart Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
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RNA was extracted with RNeasy® Fibrous Tissue Mini kit (Qiagen, Manchester, UK) using ~30 mg of snap-frozen heart tissue from the saline and DOX high group of cohort 1. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the 2200 (Agilent, RNA ScreenTape). RIN estimates for all samples were above 7. Input material was normalized to equal input of 100 ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA kit (NEB) following manufacturer’s instructions. Libraries were amplified on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (based on 10.1186/1472-6750-13-104). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 or 4200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to loading on the sequencer. Paired end sequencing was performed using a HiSeq4000 75 bp platform (Illumina, HiSeq 3000/4000 PE Cluster Kit and 150 cycle SBS Kit), generating a raw read count of 22 million read pairs per sample.
3
CD4+ T cell RNA-seq Protocol
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CD4+ T cells were isolated using negative selection (Stem Cell Technologies CD4 enrichment kit) according to the manufacturer's recommendations, and samples were processed in one batch. Total RNA was extracted using the Qiagen RNA Kit with Qiashredder columns and was sent for library preparation and sequencing. Material was quantified using RiboGreen (Invitrogen) on the FLUOstar OPTIMA plate reader (BMG Labtech) and the size profile and integrity analysed on the 2200 TapeStation (Agilent, RNA ScreenTape). Input material was normalised to 100ng prior to library preparation. Polyadenylated transcript enrichment and strand specific library preparation was completed using NEBNext Ultra II mRNA Kit (NEB) following manufacturer's instructions. Libraries were amplified on a Tetrad (Bio-Rad) using in-house unique dual indexing primers (Lamble et al., 2013) (link). Individual libraries were normalised using Qubit, and the size profile was analysed on the 2200 TapeStation. Individual libraries were normalised and pooled together accordingly. The pooled library was diluted to ~10 nM for storage. The 10 nM library was denatured and further diluted prior to sequencing.
Paired end sequencing was performed using an Illumina Novaseq6000 platform at 150 paired end mode (Illumina, San Diego, CA).
Paired end sequencing was performed using an Illumina Novaseq6000 platform at 150 paired end mode (Illumina, San Diego, CA).
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