For Figure 1 A,B, Legacy Ribo-Zero (Illumina), RiboCop (Lexogen), and NEBNext (NEB) depletions were performed after linker ligation according to the manufacturer's recommendations. For Ribo-Zero depletion, we omit the final heating step in the manufacturer's protocol, as this has been suggested to improve depletion of small fragments (McGlincy and Ingolia 2017 (link)). For subsequent figures NEBNext and Ribo-Zero plus (Illumina) depletion were performed right after fragment size selection, while all other methods were performed after linker ligation. These methods were performed according to manufacturer recommendations, except that for some experiments with Ribo-Zero Plus we included 45% formamide in the hybridization reaction (indicated as F), as well as supplementary oligos (indicated as O), provided by Illumina, designed against abundant rRNA contaminants.
Ribo zero
Manufactured by Illumina
Sourced in United States, United Kingdom
Ribo-Zero is a lab equipment product designed for the removal of ribosomal RNA (rRNA) from total RNA samples. It facilitates the enrichment of non-coding RNA species, such as messenger RNA (mRNA), for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (26 mentions)
Trizol, by Thermo Fisher Scientific (21 mentions)
Hiseq 2000, by Illumina (18 mentions)
Hiseq 4000, by Illumina (11 mentions)
Trizol reagent, by Thermo Fisher Scientific (10 mentions)
Turbo dnase, by Thermo Fisher Scientific (10 mentions)
Rneasy mini kit, by Qiagen (10 mentions)
Nextseq 500, by Illumina (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (8 mentions)
Rneasy kit, by Qiagen (8 mentions)
Truseq stranded total rna library prep kit, by Illumina (8 mentions)
Hiseq 2000 sequencer, by Illumina (6 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Bioanalyzer, by Agilent Technologies (5 mentions)
Nextseq 500 device, by Illumina (5 mentions)
Dnase, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions)
187 protocols using ribo zero
1
Comprehensive rRNA Depletion Techniques
2
Comprehensive RNA-seq Library Preparation
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Total RNA was extracted and rRNA was removed using the Epicenter Ribo-ZeroTM (Epicentre, Madison, WI, USA) following the manufacturer’s procedure. The total RNA quantity and purity were analyzed using Aglient 2100 bioanalyzer test (the RIN value of total RNA was ≥7.0, 28S/18S ≥ 1.0) (Agilent, Santa Clara, CA, USA), Qubit 2.0 detection (total RNA concentration ≥ 65 ng/uL) and Nanodrop detection (OD260/280 ≥ 1.8, OD260/230 ≥ 0.5).
Epicentre epicentre Ribo-ZeroTM kit was used to remove rRNA; rRNA-depleted RNA was interrupted randomly by Fragmentation Buffer; random hexamer primer was used to synthesize, first, cDNA; then, second, cDNA was synthesized; cDNA was purified by AMPure XP beads; the purified double-stranded cDNA was repaired; A was added and sequenced; AMPure XP Beads were used for fragment size selection; the U chain was degraded; and then, the cDNA library was enriched by PCR. After the construction of the library, the concentration and insert size of the library was detected using Qubit 2.0 and Agilent 2100, respectively. The effective concentration of the library was accurately quantified by the Q-PCR method so as to ensure the library quality (determined to be >2 nM). After detection, different libraries were pooled based on the target machine data volume, and were sequenced on the Illumina Hi-Seq platform of BioMarker Technologies (Beijing, China).
Epicentre epicentre Ribo-ZeroTM kit was used to remove rRNA; rRNA-depleted RNA was interrupted randomly by Fragmentation Buffer; random hexamer primer was used to synthesize, first, cDNA; then, second, cDNA was synthesized; cDNA was purified by AMPure XP beads; the purified double-stranded cDNA was repaired; A was added and sequenced; AMPure XP Beads were used for fragment size selection; the U chain was degraded; and then, the cDNA library was enriched by PCR. After the construction of the library, the concentration and insert size of the library was detected using Qubit 2.0 and Agilent 2100, respectively. The effective concentration of the library was accurately quantified by the Q-PCR method so as to ensure the library quality (determined to be >2 nM). After detection, different libraries were pooled based on the target machine data volume, and were sequenced on the Illumina Hi-Seq platform of BioMarker Technologies (Beijing, China).
3
Total RNA Extraction and Purification
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Total RNA was isolated from each sample using the plant RNA isolation kit (Aidlab Company, Beijing, China) according to the manufacturer’s instructions. rRNA was removed using the Epicenter Ribo-ZeroTM (Epicentre, Madison, WI, USA) following the manufacturer’s procedure. RNA degradation and contamination, especially DNA contamination, were monitored on 1.5% agarose gels. The RNA concentration and purity were measured by using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). The RNA integrity was assessed by using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA).
4
Total RNA Extraction and Strand-Specific RNA-Seq
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Total RNA was collected from organoid cultures by TRIzol (Thermo Fisher) extraction and depleted of rRNA by Ribo-Zero (Epicentre). Bulk strand-specific total-transcriptome RNA-seq libraries were prepared using dUTP during second-strand synthesis either with the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) or with home brew components (Parkhomchuk et al., 2009 (link)).
5
RNA-Seq Analysis of IL-1β-Induced Chondrocytes
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Primary hip OA chondrocytes (n = 3 patients) were left unstimulated or stimulated with IL‐1β (1 ng/ml) for 4 hours in 0.1% FCS culture media in the absence of antibiotics and amphotericin. Total RNA was isolated using TRIzol reagent (Life Technologies), further purified (RNeasy column; Qiagen), and the RNA integrity number (RIN) was assessed (Agilent Bioanalyzer). All RIN values were >7, and 260:280 ratios (measured by NanoDrop) were >1.7. Ribosomal RNA was removed using Ribozero (Epicentre Technologies), and RNAseq (100‐bp paired‐end, stranded sequencing) was performed on an Illumina HiSeq 2000 sequencer. Subsequent analysis was undertaken using Tophat2/Cufflinks with alignment against the hg19 reference genome (Figure 1 A). LncRNAs were identified using Cufflinks and then compared with known lncRNAs previously annotated in Gencode version 19 and the Human LincRNAs Catalog 29 . CuffDiff was used to compare control and IL‐1β–treated cells to identify differentially expressed transcripts (false discovery rate [FDR] <0.05, fold change >2, and change in fragments per kilobase of transcript per million mapped reads [FPKM] >1). Sequence data are available through the GEO database under series number GSE74220.
6
RNA Extraction and ERCC Spike-In
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Commercial total RNA samples were obtained for 4 different adult human (Ambion AM6000) and mouse (Clontech 636644) tissues: heart, testes, liver and brain. From the same panel also came mouse E7 and E15 samples. Human K562 and HeLa RNA was obtained directly from members of the ENCODE consortium31 (link). Neither cell line used in this paper is listed in the database of commonly misidentified cell lines maintained by ICLAC. Cell lines were not authenticated. Cell lines were tested for mycoplasma contamination as per ENCODE guidelines (see URLs). The integrity of samples were tested by Bionanalyzer (Agilent) and all had values of 8.5 or higher. To 4 µg of each RNA sample, we added 4 µl of 1:100 diluted ERCC mix (Ambion 4456740) according to manufacturer’s protocol (Supplementary Table 6 ). Mix 1&2 were assigned to samples as shown below. The samples containing ERCC controls were ribodepleted with Ribo-Zero (Epicentre MRZE724), and successful rRNA removal was validated by Bioanalyzer.
7
Comprehensive RNA-seq and PacBio Sequencing of MCF-7 Breast Cancer Cell Line
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MCF-7 is one of the most commonly used breast cancer cell lines. We used both short-read and long-read data for this sample in our analyses.
Illumina MiSeq/HiSeq sequencing. Prior to sequencing, the MCF7 total RNA was assessed for fragmentation and quality using Agilent Bioanalysis and Qubit, respectively. RNA-seq libraries were prepared after ribosomal depletion, using Epicentre RiboZero commercial reagents. Following cDNA preparation, Covaris shearing was conducted to an insert size of ~600 bp as assessed by Agilent Bioanalysis using standard Illumina adapters and PCR cycle conditions for sequencing on the Illumina MiSeq instrument. 2 × 300 bp paired-end sequencing was then conducted across two Illumina MiSeq flow cell lanes using version 3 commercial kits to assure the longest read length possible. An additional Illumina HiSeq lane was completed on the Illumina HiSeq 2500 with 2 × 100 bp paired-end reads totaling ~127 M additional reads with ~83% over QV30. All data were then merged for analysis.
PacBio sequencing. The PacBio sequences for MCF-7 were obtained from the PacBio’s 2013 release of the MCF7 transcriptome data (http://www.pacb.com/blog/data-release-human-mcf-7-transcriptome/ ).
Illumina MiSeq/HiSeq sequencing. Prior to sequencing, the MCF7 total RNA was assessed for fragmentation and quality using Agilent Bioanalysis and Qubit, respectively. RNA-seq libraries were prepared after ribosomal depletion, using Epicentre RiboZero commercial reagents. Following cDNA preparation, Covaris shearing was conducted to an insert size of ~600 bp as assessed by Agilent Bioanalysis using standard Illumina adapters and PCR cycle conditions for sequencing on the Illumina MiSeq instrument. 2 × 300 bp paired-end sequencing was then conducted across two Illumina MiSeq flow cell lanes using version 3 commercial kits to assure the longest read length possible. An additional Illumina HiSeq lane was completed on the Illumina HiSeq 2500 with 2 × 100 bp paired-end reads totaling ~127 M additional reads with ~83% over QV30. All data were then merged for analysis.
PacBio sequencing. The PacBio sequences for MCF-7 were obtained from the PacBio’s 2013 release of the MCF7 transcriptome data (
8
Mitochondrial RNA Sequencing of CMS Flower Buds
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The extracted mitochondrial RNA from the flower buds (3–5 mm size) in CMS line 2074A, its maintainer 2074B and fertile material F1 (2074A × E5903) were sequenced on an Illumina HiSeq2000 at BGI (Beijing Genomics Institute). Ribosomal RNAs were removed from the extracted mitochondrial RNA using Ribo-Zero (Epicentre, Madison, WI) and the mitochondrial RNA libraries were prepared using Illumina’s TruSeq RNAseq Sample Prep kit. Libraries were sequenced on one lane with 4 Gb clean reads/samples of an average length of 90 nt for paired-end. RNA sequence data quality was checked using FastQC to remove the adapters, low-quality, containing N bases and short sequences with reads Q30 > 85%. The reads were mapped to the assembled mitogenome of CMS line 2074A using bowtie2 [54 (link)] with the following parameters: -D 5 -R 1 -N 0 -L 25 -i S, 1, 2.00. Then, the resulting SAM files from bowtie2 mapping were transformed into BAM files using samtools view program [45 (link)]. The bowtie2 mapping results of these pair-end reads in BAM files were then used to calculate read count for each gene through HTSeq-count program [55 (link)]. Differentially expressed genes that showed up and down regulation between samples were defined based on the standards of cutoff: two-fold change and a p-value of less than 0.05.
9
Xenopus RNA-seq and Circular RNA Analysis
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Samples were depleted of rRNA using Ribozero or Ribozero-Gold kits (Epicenter) according to the standard protocol. Subsequently, libraries were prepared using the TruSeq RNA sample preparation protocol (Illumina) or TruSeq stranded total RNA sample preparation (Illumina). Sequencing was performed on an Illumina HiSeq 2000 sequencer with either 50- or 100-bp single-end reads. Reads were aligned to the X. tropicalis genome (version 4.1) or the X. laevis genome (version 6.0) using the TopHat (version 2.0.7) and Bowtie (version 2.0.6.0) sequence alignment programs (Langmead et al. 2009 (link); Trapnell et al. 2009 (link)). Exonic and intronic regions were quantified using BEDtools version 2.15.0 and Cufflinks version 2.0.2 (Quinlan and Hall 2010 (link); Trapnell et al. 2010 (link)). Features with FPKM ≥2 were considered expressed. Sequence alignments were examined in the Integrative Genomics Viewer (IGV) from the Broad Institute (Robinson et al. 2011 (link)). To search for lariats or other circular RNA reads, we ran the PERL script findlariat.pl (Taggart et al. 2012 (link)). Further analysis of sisRNAs was done using BEDtools in conjunction with custom PERL scripts.
10
Transcriptome Profiling of Malaria Mosquito
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Stage 4 larvae and 3- to 5-day-old naïve males and virgin nonblood-fed females from homokaryotypic colonies were frozen in TRIzol (Invitrogen) for subsequent total RNA extraction. Specimens were macerated using motorized pestles and the resulting total RNA samples were purified with RNeasy Mini kit (Qiagen). Concentration, quality, and integrity of the RNA samples were assessed using a Qubit 2.0 Fluorometer, a NanoDrop 8000 Spectrophotometer, and with the RNA 6000 Pico kit in a BioAnalyzer 2100 (Agilent Technologies), respectively. Unstranded (female) and stranded (male and larvae), non-polyA, ribodepleted libraries were prepared using the TruSeq Total RNA (Illumina) and the Ribo-Zero (Epicentre) kits. cDNAs were sequenced using 100-bp paired-end reads on an Illumina HiSeq 2500. Size selection was performed using 2% agarose gels on the Sage Sciences Blue Pippin, resulting in a library insert size of ∼480 nt. Three biological replicates were sequenced per biological group, that is, a particular sample type by population combination, being 27 the total number of libraries (i.e., 3 samples × 3 populations × 3 replicates). The set of nine libraries corresponding to a particular sample type was sequenced jointly in two lanes.
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