The whole full thickness rumen wall samples were shipped to CSIRO’s FD McMaster Laboratory at Chiswick, Armidale, Australia and transferred into RNALater®-ICE Frozen Tissue Transition Solution (Ambion®) and stored at −20 °C. RNA was extracted from the full thickness of ~200 mg of ventral rumen tissue using the Qiagen RNeasy® Midi Kit with on-column DNase digestion. The average RNA concentration measured using a Quant-iT™ RiboGreen® RNA reagent and kit (Invitrogen™) on a fluorescent plate reader (excitation 485 nm and emission 538 nm) was 333.9 ± 143.4 (SD) ng/μl. Samples of ~4 μg of total RNA were rRNA depleted using the Ribo-Zero™ Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre®) and purified using a Qiagen RNeasy® MinElute Cleanup Kit. 12 μl rRNA depleted RNA was sent to the Ramaciotti Genomics Centre, The University of New South Wales, Australia. RNA Quality was checked and stranded libraries were prepared using SureSelect™ stranded RNA sample preparation kit (Agilent Technologies) at the sequencing centre. RNA sequencing was performed as 100 base paired-end (PE) reads in two lanes of an Illumina HiSeq2000 instrument housed at the Ramaciotti Genomics Centre. Total reads per sample ranged from 15–20 (single reads) or 30–40 million (PE) per sample. RNA sequencing results were checked for quality using FastQC at the sequencing center.
Ribo zero magnetic gold kit human mouse rat
Manufactured by Illumina
Sourced in United States
The Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) is a tool designed for the removal of ribosomal RNA (rRNA) from total RNA samples. It employs magnetic beads coated with rRNA-specific probes to selectively deplete rRNA, allowing for the enrichment of non-rRNA species such as messenger RNA (mRNA) and noncoding RNAs. This kit is suitable for use with human, mouse, and rat RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (2 mentions)
Kapa stranded rna seq library prep kit, by Illumina (2 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (2 mentions)
Hiseq 2500 system, by Illumina (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Scriptseq v2 rna seq library preparation kit, by Illumina (2 mentions)
Rnalater ice frozen tissue transition solution, by Thermo Fisher Scientific (1 mentions)
Quant it ribogreen rna reagent and kit, by Thermo Fisher Scientific (1 mentions)
Rneasy midi kit, by Qiagen (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Baseline zero dnase, by Illumina (1 mentions)
Hiseq x10 platform, by Illumina (1 mentions)
Truseq sr cluster kit v3 cbot hs, by Illumina (1 mentions)
Ammonium hydroxide, by Merck Group (1 mentions)
Acetonitrile, by Avantor (1 mentions)
Ammonium acetate, by Merck Group (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
15 protocols using ribo zero magnetic gold kit human mouse rat
1
Rumen Tissue RNA Sequencing Protocol
2
RNA-Seq Library Preparation Protocol
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Approximately 1–2 μg total RNA from each sample was used for library construction. The integrity of the total RNA was checked by agarose gel electrophoresis and the RNA concentration was quantified with a Nanodrop ND 1000 spectrophotometer (NanoDrop Technologies, United States). mRNA was enriched by NEBNext ® Poly(A) mRNA Magnetic Isolation Module (NEB, United States) and ribosomal RNA (rRNA) was depleted using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, United States). cDNA libraries were prepared using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina) according to the manufacturer’s instructions. The constructed libraries were qualified by Agilent 2100 Bioanalyzer system (Agilent Technologies, CA, United States) and quantified by qPCR.
3
WBPH S9-2 Expression Profile RNA-Seq
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The 12-day infected male and female WBPH were divided into 21 groups according to the expression level of S9-2 (Fig. 2A,B ). About 10 μg of total RNA from each sample was treated with Baseline-ZERO™ DNase (Epicentre). RiboMinus RNA was isolated using the Ribo-Zero™ Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre) using 4 additional probes targeting the ribosomal RNA of WBPH (5′-biotin-AAGCGACGTCGCTATGAACGCTTGGCCGCCACAAG-3′, 5′-biotin-ATCCATTCATGCGCGTCACTAATTAGATGACGAGG-3′, 5′-biotin-CTTCGGCAGGTGAGTTGTTACACACTCCTTAGCGG-3′, 5′-biotin-TACCGCGGCTGCTGGCACCAGACTTGCCCTCCAAT-3′) according to the kit instructions, and the libraries were then constructed according to previously described methods56 (link) and sequenced using the HiSeq X10 platform (Illumina, San Diego, CA, USA).
4
Rat Transcriptome Profiling by RNA-Seq
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Healthy male Wistar rats, SPF grade, purchased from Animal Experimental Center, Basic Medical College of Jilin University, P.R. China. Liquid chromatography-mass spectrometry (LC-MS) grade ammonium acetate, ammonium hydroxide and MeOH were purchased from Sigma-Aldrich (USA). Acetonitrile was purchased from J.T.Baker (USA). KAPA Stranded RNA-Seq Library Prep Kit and TruSeq SR Cluster Kit v3-cBot-HS were purchased from Illumina (USA). NEBNext® Poly(A) mRNA Magnetic Isolation Module was purchased from NEB (USA). RiboZero Magnetic Gold Kit (Human/ Mouse/Rat) was purchased from Epicentre (USA).
5
Nematode RNA Extraction and Depletion
6
Transcriptome Analysis of Frozen Tissues
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Total RNA was extracted from frozen tissues using TRIzol (Life Technologies, Grand Island, NY, USA) following the manufacturer's instructions. The details of RNA sequencing, data processing, and statistical analysis are included in the Supplementary Materials and Methods . Briefly, RNA-seq was performed using a Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicentre, Madison, WI, USA) and a TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA) for library preparation and run on a HiSeq 2000 (Illumina, San Diego, CA, USA). Data cleaning, sequence alignment, and gene expression quantification were done sequentially using the corresponding software. Statistical analysis was performed for normalized expression levels using K-mean clustering and Wilcoxon signed-rank test.
7
Transcriptomic Profiling of Exercise-Induced Changes
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Pre- and post-training RNA samples from eight participants were randomly chosen for transcriptomic sequencing. We analyzed only samples collected at basal and 240′ time points, as alterations in mRNA expression several-fold from basal levels are typically greatest at 3–12 h after cessation of exercise [23 (link)]. Total RNA (600 ng) was first depleted in rRNA depletion using the Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat; Epicentre, WI, USA) and rRNA-depleted samples were purified using Agencourt RNAClean XP (Beckman Coulter, CA, USA). Libraries were then prepared after rRNA depletion with the TruSeq Stranded Total Sample Prep Kit (Illumina, CA, USA). Last, libraries were quality-controlled using a Bioanalyzer instrument (Agilent Technologies) and subjected to 100-bp single-end sequencing on a HiSeq 2500 system (Illumina) at the Danish National High-Throughput DNA Sequencing Center. An average depth of 36.6 million reads was generated.
8
Stranded RNA-seq Library Preparation
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RNA was extracted from cells with TRIzol reagent (Life Technologies) and 4 mg RNA was fractionated across three NEBNext Poly(A) mRNA Magnetic Isolation Module columns. The supernatant was retained as the polyA-depleted fraction (polyA À ) and the bead-bound (polyA + ) fraction eluted from beads in elution buffer/water according to manufacturer's instructions. The polyA À fraction was size-fractionated and concentrated using 3.53 AMPure RNAClean XP sam-ple preparation (New England Biolabs) and eluted with water. Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat) (Epicenter) treatment was performed on the polyA À fraction, purified, and size-fractionated with 1.73 AMPure RNAClean XP beads. Stranded RNA libraries were made using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) and multiplexed between 2-and 4-index on HiSeq 2500, 100 base pairs (bp) paired-end reads.
9
RNA Depletion and Library Preparation
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5 μg total RNA was depleted of ribosomal and transfer RNA using RiboZero Magnetic Gold Kit (Human/Mouse/Rat)(Illumina, Scoresby, VIC, Australia). Depleted RNA was purified with the Isolate II RNA micro kit (Bioline) and assessed for quality using the Bioanalyzer (Agilent). Depleted RNA was used in library preparation using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA #E7560) in accordance with the manufacturer’s instructions. NEBNext Indexed Primers for Illumina (New England Biolabs) were used to barcode samples. Ampure XP beads (Beckman Coulter, Mount Waverley, VIC, Australia) were used for size selection and all purification steps in accordance with the manufacturer’s instructions. 15 cycles of PCR were used for library amplification. Libraries were assessed for quality using a Bioanalyzer High Sensitivity DNA ChIP (Agilent).
10
Ribosome Profiling of Mammalian Cells
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Sample preparation for ribosome profiling was conducted according to the manufacturer’s specifications for the TruSeq Ribo Profile (Mammalian) Library Prep Kit (Illumina). Briefly, HEK293 cells were treated with CHX (Sigma-Aldrich, final concentration 0.1 mg/ml) for 1 min. In-dish cell lysis was performed using mammalian lysis buffer (including CHX at a concentration of 0.1 mg/ml). Then 600 μl of lysate were taken and 15 μl of RNase I (100 U/μl, Thermo Fisher Scientific) were added and the mixtures were incubated for 45 min at room temperature, followed by adding 15 μl SUPERaseIn RNase Inhibitor (Ambion, Thermo Fisher Scientific) to stop the reaction. Ribosome recovery was performed by illustra MicroSpin S-400 HR Columns (GE Healthcare) and the RPFs were purified by RNA Clean & Concentrator (Zymo Research). Ribosomal RNAs were depleted using Ribo-Zero Magnetic Gold Kit (Human/Mouse/Rat, Illumina). RPFs without ribosomal RNA were run on a 15% urea denaturing-PAGE gel, and the gel slices corresponding to 28–30 nts were excised. The RPF RNAs were eluted and precipitated followed by library construction according to the manufacturer’s protocol.
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