Mouse back skin from CR, GF, CR×CR F1, and GF×GF F1 was collected and fixed overnight in 4% paraformaldehyde (Fisher AAJ19943K2) at 4°C followed by paraffin embedding. Laser capture microdissection (LCM) was performed using the LMD 7000 system (Leica Microsystems). FFPE mouse skin was processed and cut onto a polyethylene naphthalate (PEN) slide designed for LCM processing (Leica 11505158). At least 1,000 SGs or 1,000,000 μm2 of tissue was isolated to obtain enough material for RNA extraction. SG RNA was extracted from post-LCM tissue using a Qiagen All Prep DNA/RNA FFPE Kit (Qiagen 80234). RNA concentration was measured by Qubit fluorometric quantification (ThermoFisher Qubit 2.0 Fluorometer) and RNA quality measured via BioAnalyzer (Agilent 2100 Bioanalyzer Instrument). cDNA libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit (Illumina 20040529) with IDT for Illumina RNA UD Indexes, Set A (Illumina 20040553). Libraries were assessed for cDNA quantity and library quality using Qubit and BioAnalyzer. As necessary, an extra bead wash step was performed to remove excess primer dimers in the library and purify samples further. Samples were then pooled and sequenced on a Nextseq 550 using a NextSeq 500/550 High Output Kit v2.5 (150 Cycles) (Illumina 20024907).
Stranded total rna prep with ribo zero plus kit
Manufactured by Illumina
Sourced in United States
The Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit is a laboratory equipment product designed for the preparation of stranded total RNA samples. The kit provides a method for the depletion of ribosomal RNA (rRNA) from total RNA samples, enabling the enrichment of non-coding RNA species. The core function of this product is to facilitate the preparation of stranded total RNA samples for various downstream applications, such as transcriptome analysis.
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Lab products found in correlation
Trizol ls, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna ffpe kit, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer instrument, by Agilent Technologies (1 mentions)
Aaj19943k2, by Thermo Fisher Scientific (1 mentions)
Lmd7000 system, by Leica (1 mentions)
Idt for rna ud indexes set a, by Illumina (1 mentions)
Nextseq 500 550 high output kit v2.5 150 cycles, by Illumina (1 mentions)
Qubit 2.0 fluorometer, by Agilent Technologies (1 mentions)
Biors uhplc, by Agilent Technologies (1 mentions)
Sec 5 7, by Agilent Technologies (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
6 protocols using stranded total rna prep with ribo zero plus kit
1
Laser Capture Microdissection and RNA Sequencing of Mouse Sebaceous Glands
2
Polysome Profiling by Ribosome-SEC
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Polysome fractions were collected using the fractionation by high performance liquid chromatography-based RiboMega-SEC approach as previously described [29 (link)]. Briefly, cells were lysed in a CHAPS buffer containing RNase inhibitors and the lysate separated by size exclusion chromatography on a Thermo Dionex BioRS UHPLC and Agilent SEC-5 7.8 × 300 mm HPLC column with 2000 Å pores and 5 μm particles. The samples and column were kept at 5 °C throughout analysis. Fractions were collected across the entire separation covering polysomes, monosomes and smaller cellular complexes. Total RNA was extracted from the eluted polysome fractions using TRIzol LS (ThermoFisher, cat# 10296010). Ribosomal RNA depleted library 1 μg total RNA using the Illumina Stranded Total RNA Prep with Ribo-Zero Plus Kit (Illumina), according to the manufacturer’s instructions. A total of 25 million 150 bp paired-end strand-specific reads were sequenced per sample on an Illumina HiSeq® 2500 Platform (Illumina).
3
Long Non-Coding RNA Sequencing from Chicken Embryo
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Total RNA extraction from chicken embryo visceral tissues (CEVTs) without any modification was previously described in detail in our study [12 (link)]. Thereafter, the qualified RNA samples for library construction underwent tRNA and rRNA removal treatment before cDNA library preparation to meet the specified requirements. The preliminary cDNA library was further processed by performing cDNA end repair and Ploy A tail ligation—an essential component of lncRNA—followed by PCR reaction to obtain a complete cDNA library. This was performed according to the manufacturer’s instructions, without any modification (Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit, Illumina, San Diego, CA, USA). After the purification and quality assessment of the constructed libraries, lncRNA sequencing was conducted on an Illumina NextSeq 2000 Sequencing System (Illumina, San Diego, CA, USA).
4
ATAC-seq and RNA-seq Protocol
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ATAC-seq was performed using Tagmentation Mix (Diagenode) according to the manufacturer’s protocol with some modifications detailed in SI Appendix . For RNA-seq, RNA was purified using Qiagen RNeasy Kit according to the manufacturer’s protocol. Libraries were prepared using Illumina Stranded Total RNA Prep with Ribo-Zero Plus kit according to the manufacturer’s protocol.
5
Comparative RNA-Seq Analysis of E. coli K1 Strains
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E. coli K1 E11 strains WT and ∆aroA were grown in LB broth at 37 °C overnight. The following day, bacterial cultures of the 2 strains were grown from OD = 0.08 to exponential phage. At OD = 0.600, the bacteria were harvested and washed by centrifugation, then recovered in lysis buffer from the RNA extraction kit, Bioanalysis (Valencienner, Germany) reference 740955.50. Total RNA from both strains was extracted using this kit according to the manufacturer’s instructions. RNA concentration was quantified using a spectrophotometer (Nanodrop Technologies). RNA-Seq analysis was carried out by the Genotyping/Sequencing Platform of the Brain Institute—ICM, France. mRNA library preparation was realized following manufacturer’s recommendations (Stranded Total RNA Prep with Ribo-Zero Plus Kit from ILLUMINA). Final samples pooled library prep were sequenced on Nextseq 2000 ILLUMINA P2-200 cartridge (2x400Millions of 100 bases reads) corresponding to 2x14Millions of reads per sample after demultiplexing. RNA-Seq analysis was carried out by the Genotyping/Sequencing Platform of the Institut du Cerveau/Paris Brain Institute—ICM, France. Analysis of RNA sequencing data using CLC Genomics.
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Illumina Stranded Total RNA Sequencing
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