Live species were harvested by centrifugation at 1700 g for 10 min and stored at −80°C until further treatment. The total RNA was extracted using the RNeasy kit (Qiagen, Hilden, Germany) and digested with DNase. The rRNA fraction was depleted using GeneRead rRNA Depletion Kit (Qiagen, Hilden, Germany). The quality of the remaining RNA was assayed using a BioAnalyzer (Agilent Technologies, Palo Alto, CA, USA). In total, 250 ng of the RNA was used to synthesize cDNA using Affymetrix 30 IVT Express Kit (Affymetrix Inc., Santa Clara, CA, USA). The resulting high quality cDNAs were used to construct a library for paired-end sequencing.
Generead rrna depletion kit
Manufactured by Qiagen
Sourced in Germany
The GeneRead rRNA Depletion Kit is a laboratory equipment product designed to selectively deplete ribosomal RNA (rRNA) from RNA samples, allowing for improved detection and analysis of non-ribosomal RNA species. The kit utilizes hybridization-based capture probes to remove rRNA, enabling a more comprehensive and accurate assessment of the transcriptome.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy kit, by Qiagen (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Nebnext ultra directional rna library prep kit for, by Illumina (2 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Nebnext ultra directional rna library prep kit, by New England Biolabs (1 mentions)
Agencourt ampure kit, by Beckman Coulter (1 mentions)
Fragment analyzer, by Agilent Technologies (1 mentions)
Bioanalyzer 2200, by Agilent Technologies (1 mentions)
Rnase r, by Illumina (1 mentions)
Rnaprep pure tissue kit, by Tiangen Biotech (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Kapa rna hyperprep kit with riboerase, by Roche (1 mentions)
Kapa hifi dna polymerase, by Roche (1 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Paxgene blood rna system, by Qiagen (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Truseq stranded mrna lt sample prep kit, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
14 protocols using generead rrna depletion kit
1
RNA Sequencing Library Preparation
2
RNA-Seq Library Preparation and Sequencing
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3 μg of total RNA per sample were used for library preparation. Ribosomal RNA was depleted by using the GeneRead rRNA Depletion Kit (Qiagen). RNA fragmentation, cDNA synthesis and further RNA-Seq library preparation was done with the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs). After enrichment and XP bead (Agencourt AMPure Kit; Beckman Coulter, Inc.) purification, quality control was done using Fragment Analyzer (Advanced Analytical). The bar-coded libraries were equimolarly pooled and subjected to 75 bp single-end sequencing on Illumina HiSeq 2000, resulting in an average of 33 million reads per sample. Sequencing raw data were deposited in GEO database under the GSE63948 accession number. The “Tuxedo Suite” of Bowtie, TopHat, Cufflinks and Cuffdiff [39 (link),40 (link),58 (link),59 (link)] was used for the alignment and expression analysis. We aligned the samples separately to the mm9 genome using the splice junction mapper Tophat (version 2.0.9), which used Bowtie 2 (version 2.1.0) for mapping. The Ensembl version 67 [41 (link)] was used as a support for the annotation during the alignment.
3
Total RNA Extraction and rRNA Depletion
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Total RNA was extracted from LUAD and paired nontumor tissues by TRIzol reagent (Invitrogen, Shanghai, China) separately. The RNA quality was checked by Bioanalyzer 2200 (Agilent, Santa Clara, CA, USA) and kept at −80 °C. The RNA with RIN > 8.0 are right for rRNA depletion. rRNA was depleted by GeneRead rRNA Depletion Kit (QIAGEN, Venlo, Germany).
4
Comprehensive transcriptome analysis of circRNA
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Total RNA were isolated from the cultured cells and fresh tissues by Trizol reagent (Invitrogen, USA). Total RNA was extracted by RNAprep pure Tissue Kit (TIANGEN), and rRNA was depleted by GeneRead rRNA Depletion Kit (QIAGEN). The mRNA was purified from total RNA by oligo (dT) magnetic beads, and fragmented into 200–500 bp; the cleaved RNA fragments were reverse-transcribed into cDNA, and enriched by PCR to create the final complementary DNA (cDNA) libraries. The harvested target bands were quantified by Agilent 2100 and then subjected to deep sequencing with the Illumina HiSeq 2000. HCC cells were mixed with RNase R (3 U/μg, Epicentre, Madison, WI) at 37 °C for 15 min, and then qRT-PCR was used to assess the expression stability of circ0003998 as compared to ARFGEF2 mRNA.
5
Comprehensive RNA-seq Library Preparation
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For paRNA-seq, we purified poly(A) RNA from 1 µg of ES total RNA using a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). For rdRNA-seq, we depleted rRNA from 1 µg of ES total RNA using a GeneRead rRNA Depletion Kit (Qiagen). We prepared conventional RNA-seq library DNA with the resulting RNA using a commercial kit (NEBNext Ultra Directional RNA Library Prep Kit for Illumina; NEB) in accordance with the manufacturer’s protocol, with slight modifications during RT and PCR steps. We used SuperScript III (Thermo Fisher) for RT. We used KAPA HiFi DNA polymerase (Kapa Biosystems) in the PCR step. When we performed rdRNA-seq using 10 or 1 ng of ES total RNA, we utilized a KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) for the preparation of sequencing library DNA according to the manual instructions.
6
Comprehensive RNA Extraction and Purification
7
Genomic and Transcriptomic Profiling of Freshwater Ciliates
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Six freshwater ciliate species (Chilodontopsis depressa, Trithigmostoma cucullulus, Trochilia petrani, Trochilia sp., Dysteria derouxi, and Hartmannula sinica) were collected from Xiaoxihu Lake, Qingdao, China (120.35°E, 36.06°N). For each species, cells were washed in phosphate-buffered saline (PBS) buffer (without Mg2+ or Ca2+), and genomic DNA extracted from a single cell was amplified using a Single Cell WGA Kit (Yikon, YK001A) based on MALBAC technology (Lu et al., 2012 (link)). The RNA of C. depressa and D. derouxi was extracted from a single cell using the RNeasy kit (Qiagen, Hilden, Germany) and digested with DNase. The rRNA fraction was depleted using GeneRead rRNA Depletion Kit (Qiagen, Hilden, Germany). The raw data of single-cell genome sequencing of Chilodochona sp. were supplied by Dr. Denis H. Lynn (University of British Columbia, Vancouver, Canada).
8
Ribosomal RNA Depletion and cDNA Library Preparation
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Ribosomal RNA depletion was performed using a 5′-phosphate-dependent exonuclease (GeneRead rRNA Depletion kit (Qiagen, Germany), which degraded transcripts with a 5′ monophosphate. Linear RNA depletion was performed using Ribonuclease R. The dosage ratio of Ribonuclease R to RNA is 3U:1 µg and the digestive conditions were 37°C for 30 min. According to experimental protocols, Rayscript cDNA Synthesis kit (GENEray, GK8030) was used for the reverse transcription. RNA fragments were used for sequential first-strand and second-strand cDNA synthesis, and the cDNA templates were enriched. Libraries were constructed using the TruSeq Stranded mRNA LT Sample Prep kit (Illumina, Inc., San Diego, CA, USA). Library quality was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.) prior to sequencing. Total concentrations were determined using a Qubit 12.0 Fluorometer (Thermo Fisher Scientific, Inc.). Libraries were sequenced on the Illumina NextSeq 500 platform (Illumina, Inc.) with 2×150 bp paired-end reads. The libraries were constructed and sequenced by Shanghai Personal Biotechnology Co., Ltd. (www.personalbio.cn/ ; Shanghai, China).
9
RNA Sequencing Library Preparation
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The total RNA of cells was isolated by using TRIzol and used for RNA sequencing. RNA quantification was performed with a Qubit 3.0 spectrophotometer (Thermo Fisher, MA, USA). Library preparation was performed by Epibiotek (Guangzhou, China). Briefly, total RNA was treated with the GeneRead™ rRNA Depletion Kit (Qiagen, Hilden, Germany, Cat No. 180211) to remove ribosomal RNA. rRNA-depleted RNA was fragmented and then used to construct strand-specific RNA libraries by using the VAHTS Stranded RNA-seq Library Prep Kit for Illumina (Vazyme, Nanjing, China, Cat. No NR602) according to the manufacturer’s instructions. Library quality was determined on a Qseq100 Bio-Fragment Analyzer (Bioptic, Taiwan, China). The strand-specific libraries were sequenced.
10
E2F1 RIP-Seq Analysis of Gene Regulation
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An E2F1 RIP was performed as described above, from samples treated for 72 hours with nontargeting siRNA or siRNA against TSN. An E2F1 siRNA condition was also included for the RIP-seq as a control to monitor for specificity of the RNA species identified. Following RNA extraction and DNase treatment, equal volumes of the sample were taken and underwent ribodepletion using a GeneRead rRNA Depletion kit (Qiagen). Libraries were prepared using a NEBNext Ultra Directional RNA library Prep kit for Illumina (New England Biolabs). The library was sequenced on an Illumina NextSeq, and bioinformatics analysis was carried out (see below).
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