RNAs were extracted from GEMMs prostate DLPs (5 months of age) by using the the pureLink RNA Kit (Invitrogen). Ribosomal RNA removal was carried out using RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen) according to the manufacturer’s protocol. RNAs derived from GEMMs DLPs were subjected to next-generation sequencing (NGS) to generate deep coverage RNA-seq data. For each group, sequencing was performed on three biological samples. RNA-seq libraries were prepared using Illumina TruSeq RNA Sample Prep Kit V2 and subjected to deep sequencing with Illumina HiSeq 4000 at the Molecular Biology Core Facilities at the Dana Farber Cancer Institute. To achieve comprehensive coverage for each sample, we generated ~32 million to 39 million single-end reads.
Ribominus human mouse transcriptome isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The RiboMinus Human/Mouse Transcriptome Isolation Kit is a laboratory tool designed to selectively remove ribosomal RNA (rRNA) from total RNA samples, allowing for the enrichment of messenger RNA (mRNA) and other non-coding RNAs. The kit utilizes magnetic beads coated with complementary oligonucleotides to capture and remove rRNA from the samples.
Automatically generated - may contain errors
Lab products found in correlation
Truseq rna sample prep kit v2, by Illumina (2 mentions)
Genechip human exon 1.0 st array, by Thermo Fisher Scientific (2 mentions)
Purelink rna kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 4000, by Illumina (1 mentions)
Human gene chip exon1.0 st array, by Thermo Fisher Scientific (1 mentions)
Genechip sample cleanup module, by Thermo Fisher Scientific (1 mentions)
Hg u133 plus 2.0 array, by Thermo Fisher Scientific (1 mentions)
Spoton flow cell mk 1, by Oxford Nanopore (1 mentions)
Sqk lsk108 kit, by Oxford Nanopore (1 mentions)
Poly a polymerase tailing kit, by Illumina (1 mentions)
Smarter pcr cdna synthesis kit, by Takara Bio (1 mentions)
Kapa hifi pcr kit, by Roche (1 mentions)
Trizol isolation reagent, by Thermo Fisher Scientific (1 mentions)
Genechip scanner 3000, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Quick ligation kit, by New England Biolabs (1 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (1 mentions)
8 protocols using ribominus human mouse transcriptome isolation kit
1
RNA-seq Analysis of Mouse Prostate DLPs
2
Comprehensive Transcriptomic Analysis Using Affymetrix Exon Arrays
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Total RNA was reverse-transcribed and hybridized onto Affymetrix HG-U133 Plus 2.0
array, using the identical protocol and ID names for genes as GeneChip Human Exon
1.0ST Arrays (Affymetrix, Santa Clara, CA, USA). The Affymetrix Human Gene Chip Exon
1.0 ST Array interrogates over one million exons representing over 17,868 NCBI
Reference Sequence (RefSeq) transcripts. Arrays were run using the
manufacturer's technical protocol (Affymetrix, Santa Clara, CA). Briefly, 2 qg
of total RNA was subjected to a ribosomal RNA removal procedure (RiboMinus Human/
Mouse Transcriptome Isolation Kit, Invitrogen - Thermo Fischer Scientific) to reduce
the 28S and 18S rRNA population to minimize background and increase sensitivity of
the assay. Reduced RNA was reverse-transcribed to cDNA using random hexamers tagged
with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA
polymerase (GeneChip WT cDNA Synthesis Kit, Affymetrix). The resulting
double-stranded cDNA was used for amplification of antisense cRNA and cleaned using
the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was
performed using random primers to reverse transcribe the cRNA into sense single
stranded DNA, which was fragmented, labeled, and hybridized to arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station and G7 Affymetrix
high-resolution scanner using GCOS 1.3.
array, using the identical protocol and ID names for genes as GeneChip Human Exon
1.0ST Arrays (Affymetrix, Santa Clara, CA, USA). The Affymetrix Human Gene Chip Exon
1.0 ST Array interrogates over one million exons representing over 17,868 NCBI
Reference Sequence (RefSeq) transcripts. Arrays were run using the
manufacturer's technical protocol (Affymetrix, Santa Clara, CA). Briefly, 2 qg
of total RNA was subjected to a ribosomal RNA removal procedure (RiboMinus Human/
Mouse Transcriptome Isolation Kit, Invitrogen - Thermo Fischer Scientific) to reduce
the 28S and 18S rRNA population to minimize background and increase sensitivity of
the assay. Reduced RNA was reverse-transcribed to cDNA using random hexamers tagged
with a T7 promoter sequence followed by a second strand cDNA synthesis using DNA
polymerase (GeneChip WT cDNA Synthesis Kit, Affymetrix). The resulting
double-stranded cDNA was used for amplification of antisense cRNA and cleaned using
the Gene Chip Sample Cleanup Module (Affymetrix). A second cycle cDNA synthesis was
performed using random primers to reverse transcribe the cRNA into sense single
stranded DNA, which was fragmented, labeled, and hybridized to arrays. Arrays were
washed, stained, and scanned on the Affymetrix Fluidics Station and G7 Affymetrix
high-resolution scanner using GCOS 1.3.
3
Single-cell RNA profiling of HeLa nucleoli
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Total RNA from isolated HeLa nucleoli was depleted of rRNA by the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen). RNA was converted to double stranded cDNA using random hexamer with the NEBNext Ultra RNA First Strand and NEBNext Ultra RNA 2nd Strand Synthesis Kits (NEB). 1D library was prepared with the SQK-LSK108 kit (Oxford Nanopore) and sequenced on a SpotON Flowcell MK I (R9.4) flowcell for 14 hr using a MinION MK 1B sequencer. The flowcell was washed and another identical library was loaded in the same flowcell and sequenced for another 14 hr. Basecalling was performed with Albacore 2.0.2.
The PacBio Iso-seq and nanopore RNA-seq data sets are deposited to the NCBI SR data base. The Bioproject accession number is SRA data: PRJNA814414.
The PacBio Iso-seq and nanopore RNA-seq data sets are deposited to the NCBI SR data base. The Bioproject accession number is SRA data: PRJNA814414.
4
Sequencing of Enriched SNUL-1 Transcripts
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Total RNA from isolated HeLa nucleoli was poly-adenylated by Poly(A) Polymerase Tailing Kit (Epicentre) and depleted of rRNA by the RiboMinus Human/Mouse Transcriptome Isolation Kit (Invitrogen). RNA was then reverse transcribed by the SMARTer PCR cDNA Synthesis Kit (Clontech) and amplified for 15 cycles using KAPA HiFi PCR Kit (KAPA biosystems). cDNA was then separated into two fractions by size using 0.5 X and 1 X AMPure PB Beads (Pacific Scientific), respectively. SNUL-1 was then enriched from the two fractions by xGen capture procedure with SNUL-1 Probe 4 using the xGen hybridization and Wash Kit (IDT). Another round of PCR amplification was carried out after the capture. The two fractions were then combined. Library was prepared by Amplicon SMRTbell Prep (Pacific Scientific) and sequenced on LR SMRT cell with 20 hr movie.
5
Affymetrix Exon Array Gene Expression Analysis
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After 48 h of incubation (the same time as that used for the RILA assay), total RNA was isolated using TRIzol isolation reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MS, USA); see Supplementary Information
6
Identification and Classification of RNA Binding Proteins
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Potential RNA binding proteins (RBPs) from high throughput screening of HeLa reporter cells were identified by criteria listed in Figure 1E and DAVID Bioinformatics Resources (v6.7) (Huang da et al., 2009) . Further classification of 103 RBPs was manually clustered with UniProt Knowledgebase (UniProt, 2017) (http://www.uniprot.org/).
Polyadenylated/Non-polyadenylated RNA separation, Ribosomal RNA depletion and RNA-seq Polyadenylated and non-polyadenylated RNA separation was carried out as described (Yang et al., 2011; Yin et al., 2015) . Nascent RNA purification was carried out as described (Zhang et al., 2016b) . Briefly, prior to construct non-polyadenylated or nascent RNAseq library, rRNA was depleted as described (Yang et al., 2011) . Ribosomal RNA removal was carried out using RiboMinus Human/ Mouse Transcriptome Isolation Kit (Invitrogen) according to the manufacturer's protocol. RNA-seq libraries were prepared using Illumina TruSeq RNA Sample Prep Kit V2 and subjected to deep sequencing with Illumina HiSeq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.
Polyadenylated/Non-polyadenylated RNA separation, Ribosomal RNA depletion and RNA-seq Polyadenylated and non-polyadenylated RNA separation was carried out as described (Yang et al., 2011; Yin et al., 2015) . Nascent RNA purification was carried out as described (Zhang et al., 2016b) . Briefly, prior to construct non-polyadenylated or nascent RNAseq library, rRNA was depleted as described (Yang et al., 2011) . Ribosomal RNA removal was carried out using RiboMinus Human/ Mouse Transcriptome Isolation Kit (Invitrogen) according to the manufacturer's protocol. RNA-seq libraries were prepared using Illumina TruSeq RNA Sample Prep Kit V2 and subjected to deep sequencing with Illumina HiSeq 2500 at CAS-MPG Partner Institute for Computational Biology Omics Core, Shanghai, China.
7
RNA Sequencing Library Preparation
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Ribosomal RNAs (rRNAs) were removed from 1 μg total RNA using the RiboMinus™ Transcriptome Isolation kit (Human/Mouse) (Invitrogen) according to the manufacturer’s instructions. The remaining RNA was heat fragmented at 94 °C for 5 min in 5 × Array Fragmentation Buffer (Ambion). The fragmented RNA was reverse transcribed to cDNA using SuperScript Double Stranded cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA). The resulting cDNA was purified using QiaQuick PCR column (Qiagen, Valencia, CA, USA), and the ends were ligated with adapters using the Quick Ligation™ Kit (New England Biolabs, Ipswich, MA, USA).
8
Postnatal Testis Transcriptome Isolation
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C57BJ/6 male mice were purchased from WeiTongLiHua experimental animal technical company (Beijing, China) and euthanized by cervical dislocation. Freshly dissected tissues were immediately frozen with liquid nitrogen, ground into powder, and immediately homogenized using TRIzol® Reagent (Ambion) for total RNA isolation. Mice of 3- and 4-weeks old were chosen for postnatal testis development studies. All procedures have been approved by the Institutional Animal Care and Use Committee (IACUC) of the Beijing Institute of Genomics. Enrichment of mRNA from total RNA was performed using Dynabeads® mRNA Purification Kit (Ambion). For rRNA depletion, the purified mRNA was further treated using Ribominus Transcriptome Isolation Kit (Human/Mouse) (Invitrogen).
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