For SARS-CoV-2 genome sequencing, 200 μL of supernatant containing virus was collected and added to 600 μL TRK lysis buffer plus beta-mercaptoethanol (BME) according to the E.Z.N.A. Total RNA kit I (Omega Bio-Tek). Total RNA was extracted according to the manufacturer’s protocol and eluted into RNase/DNase-free H2O and used for library construction. rRNA was removed from total RNA by Ribo-Zero depletion (Illumina). Indexed sequencing libraries were prepared using TruSeq RNA library preparation kit (Illumina), pooled, and then sequenced using the Illumina NextSeq system. The raw sequence data were analyzed using the LoFreq pipeline to call the mutations in the entire virus genome (42 (link)). In brief, Illumina sequencing fastq data were aligned by BWA with the SARS-CoV-2 reference genome sequence (GenBank accession no. NC_045512 ) after indexing to generate the aligned SAM and BAM files. The read group was added by Picard after the aligned BAM file sorting and indexing with SAMtools, and then duplicates were removed by Picard MarkDuplicates. Local realignment was achieved by GATK3, and then variants were called by LoFreq to generate the mutant report file.
Ribo zero depletion
Manufactured by Illumina
Sourced in United States
Ribo-Zero depletion is a lab equipment product designed to remove ribosomal RNA (rRNA) from RNA samples. It is a crucial tool for researchers studying gene expression, as it allows them to focus on the analysis of messenger RNA (mRNA) and other non-rRNA species, which are the primary targets of interest in many transcriptomics applications.
Automatically generated - may contain errors
Lab products found in correlation
Total rna kit 1, by Omega Bio-Tek (2 mentions)
Truseq rna library preparation kit, by Illumina (2 mentions)
Nextseq system, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Model 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Sequencing adapter, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Solexa genome analyzer 2, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Truseq stranded total rna sample preparation kit, by Illumina (1 mentions)
Nanodrop nd 1000 spectrometer, by Thermo Fisher Scientific (1 mentions)
Zymo dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Rneasy plus mini, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
Bioanalyzer, by Illumina (1 mentions)
6 protocols using ribo zero depletion
1
SARS-CoV-2 Genome Sequencing Protocol
2
Transcriptomic Analysis of Pregnant Uterine Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Endometrial tissue samples from the pregnant uteri of baboon (n = 3), mouse (n = 3), hamster (n = 3), bat (n = 2), and squirrel (n = 2) were dissected and mailed to the University of Chicago in RNA-Later. These samples were further dissected to remove myometrium, luminal epithelium, and extra-embryonic tissues, and then washed three times in ice cold PBS to remove unattached cell debris and red blood cells. Total RNA was extracted from the remaining tissue using the RNeasy Plus Mini Kit (74134, QIAGEN) per manufacturer’s instructions. RNA concentrations were determined by Nanodrop 2000 (Thermo Scientific). A total amount of 2.5 μg of total RNA per sample was submitted to the University of Chicago Genomics Facility for Illumina Next Gen RNA sequencing. Quality was assessed with the Bioanalyzer 2100 (Agilent). A total RNA library was generated using the TruSEQ stranded mRNA with RiboZero depletion (Illumina) for each sample. The samples were fitted with one of six different adapters with a different 6-base barcode for multiplexing. Completed libraries were run on an Illumina HiSEQ2500 with v4 chemistry on two replicate lanes for hamster and one lane for other species of an eight lane flow cell, generating 30–50 million 50 bp single-end reads per sample.
3
RNA Extraction and RNA-seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from each human tissue sample by the use of TRIzol (Invitrogen, CA, USA) according to the instructions provided by the manufacturer. Total RNA quality and quantity were verified by the use of a model 2100 Bioanalyzer (Agilent Technologies, CA, USA) and a NanoDrop ND-1000 spectrometer (Thermo Scientific, DE, USA), respectively.
RNA-seq 1 was performed as described previously (42 (link), 43 (link)). Briefly, polyadenylated [poly(A)+] RNA was obtained with two rounds of poly(A) selection using Dynabeads Oligo(dT)25 (Invitrogen). DeLi-Seq was used to prepare the sequencing library and then purified by the use of a Zymo DNA Clean and Concentrator-5 kit (Irvine, CA). The products (∼200 to ∼300 bp) were sequenced using Illumina/Solexa Genome Analyzer II.
For RNA-seq 2, libraries were prepared using a TruSeq stranded total RNA sample preparation kit with Ribo-Zero depletion (Illumina, CA, USA). In brief, high quality total RNA (1 µg) was subjected to Ribo-Zero depletion, fragmented, and then subjected to reverse transcription (RT). Double-stranded cDNAs were A-tailed and ligated with Illumina sequencing adapters. Subsequently, the ligated products were enriched by PCR and size-selected by agarose gel electrophoresis. The products (∼200 to ∼400 bp) were sequenced by the use of an Illumina Hi-seq 2500 platform with a paired 50-mer sequencing modality.
RNA-seq 1 was performed as described previously (42 (link), 43 (link)). Briefly, polyadenylated [poly(A)+] RNA was obtained with two rounds of poly(A) selection using Dynabeads Oligo(dT)25 (Invitrogen). DeLi-Seq was used to prepare the sequencing library and then purified by the use of a Zymo DNA Clean and Concentrator-5 kit (Irvine, CA). The products (∼200 to ∼300 bp) were sequenced using Illumina/Solexa Genome Analyzer II.
For RNA-seq 2, libraries were prepared using a TruSeq stranded total RNA sample preparation kit with Ribo-Zero depletion (Illumina, CA, USA). In brief, high quality total RNA (1 µg) was subjected to Ribo-Zero depletion, fragmented, and then subjected to reverse transcription (RT). Double-stranded cDNAs were A-tailed and ligated with Illumina sequencing adapters. Subsequently, the ligated products were enriched by PCR and size-selected by agarose gel electrophoresis. The products (∼200 to ∼400 bp) were sequenced by the use of an Illumina Hi-seq 2500 platform with a paired 50-mer sequencing modality.
4
RNA-seq analysis of endothelial cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HUVEC or FACS-sorted zebrafish ECs were lysed with RLT buffer containing β-mercaptoethanol and RNA was isolated with the RNeasy Plus Mini or Micro Kit (Qiagen), respectively. RNA was quantified with NanoDrop (Thermo Fisher Scientific) and quality assessment was performed with a Bioanalyzer (Agilent). Libraries were prepared using TruSeq Stranded Total RNA Sample Prep with RiboZero depletion (Illumina), quantified with Qubit (Thermo Fisher Scientific), analyzed for fragment distribution with a Bioanalyzer and sequenced with paired-end settings (2 × 75 cycles) on a MiSeq System (Illumina).
5
SARS-CoV-2 Genome Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For SARS-CoV-2 genome sequencing, 200 μL of supernatant containing virus was collected and added to 600 μL TRK lysis buffer plus beta-mecaptoethanol (BME) according to the E.Z.N.A. Total RNA Kit I (Omega Bio-tek). Total RNA was extracted according to the manufacturer’s protocol and eluted into RNase/DNase free H2O and used for library construction. Ribosomal RNA was removed from total RNA by Ribo-Zero depletion (Illumina). Indexed sequencing libraries were prepared using TruSeq RNA library preparation kit (Illumina), pooled and then sequenced using Illumina NextSeq system. The raw sequence data were analyzed using the LoFreq pipeline to call the mutations in the entire virus genome 41 (link). In brief, Illumina sequencing fastq data was aligned by BWA with the SARS-CoV-2 reference genome sequence (NC_045512.2) after indexing to generate the aligned sam and bam files. Read group was added by Picard after the aligned bam file sorting and indexing with SAMtools, and then duplicates were removed by Picard MarkDuplicates. Local realignment was achieved by gatk3 and then variant was called by LoFreq to generate the mutant report file.
6
Comparative RNA-seq Analysis of ES Cells and Embryos
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA-seq was performed on three independent cultures of ES cell lines and three single WT and E2f6−/− embryos collected from the same litters. RNAs from ESCs were extracted using the RNeasy kit (Qiagen). For embryos, we simultaneously prepared genomic DNA and total RNA from whole single embryos with the AllPrep DNA/RNA Mini Kit (Qiagen). RNA quality was verified on a 2100 Bioanalyzer (Agilent). RNA-seq libraries were generated from 250 ng of total RNA using the TruSeq Stranded Total RNA Library Prep kit with Ribo-Zero depletion (Illumina) according to the manufacturer’s instructions, and sequenced in paired-end 2 × 100 bp on an Illumina HiSeq4000. Reads were mapped using TopHat v2.0.13 with a RefSeq transcriptome index and counted in RefSeq genes with HTSeq v0.7.2 (parameters –t exon –s reverse). Differentially expressed genes were identified using DESeq2 (fold change >3, adjusted p-value < 0.0001). Genes from the Y chromosome were excluded. GO analysis was performed with DAVID 6.8 (https://david.ncifcrf.gov ). We also used published RNA-seq datasets, which are described in the “Data availability” statement.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!