We did not have access to viably preserved peripheral blood mononuclear cells (PBMCs) amenable to flow cytometry for most patients but did have frozen plasma depleted whole blood (PDWB) containing leukocyte RNA for the majority of our cohort. Leukocyte RNA was extracted from either PDWB or PBMCs. For PDWB, RNA was extracted from 200μL of PDWB by mixing with 600μL TRIzol LS Reagent (ThermoFisher), 200μL of chloroform was used for phase separation. For PBMCs, pellets were lysed with 600μL of TRIzol Reagent and 150μL of chloroform was used for phase separation. For both starting materials, the aqueous phase from the initial phase separation was then mixed with equal volume of 100% ethanol, then loaded onto columns for the RNeasy Micro Kit (Qiagen) and the protocol was followed per manufacturer’s instruction, including on-column DNase treatment. The GLOBINclear Kit (ThermoFisher) was utilized to deplete globin mRNA per the manufacturer’s instructions in the PDWB samples. Before proceeding to RNA-seq, RNA was quantified using the Quant-iT RiboGreen RNA Assay Kit (ThermoFisher) and quality was confirmed by DV200 and RIN after RNA Pico Bioanalyzer Analysis. All RNA samples sequenced had DV200 ≥ 80%.
Globinclear kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The GLOBINclear kit is a laboratory product designed to remove globin mRNA from total RNA samples. It is a tool used in the preparation of samples for subsequent analysis, such as microarray or RNA sequencing experiments.
Automatically generated - may contain errors
Lab products found in correlation
Paxgene blood rna kit, by Qiagen (10 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (9 mentions)
Tempus spin rna isolation kit, by Thermo Fisher Scientific (8 mentions)
Bioanalyzer, by Agilent Technologies (6 mentions)
Nanodrop 1000 spectrometer, by Thermo Fisher Scientific (6 mentions)
Paxgene blood mirna kit, by Qiagen (5 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Quant it ribogreen rna assay kit, by Thermo Fisher Scientific (4 mentions)
Agilent bioanalyzer, by Agilent Technologies (4 mentions)
Hiseq 2000, by Illumina (4 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (4 mentions)
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500 v4 system, by Illumina (3 mentions)
Paxgene blood rna tube, by BD (3 mentions)
Humanht 12 v4 expression beadchip, by Illumina (3 mentions)
Truseq rna sample preparation kit v2, by Illumina (3 mentions)
Quant it ribogreen assay, by Thermo Fisher Scientific (3 mentions)
Agilent 2100 bioanalyzer rna 6000 pico chip, by Agilent Technologies (3 mentions)
Totalprep rna amplification kit, by Illumina (3 mentions)
61 protocols using globinclear kit
1
High-quality RNA extraction from PBMC and PDWB
2
Assessing Gene Expression in Radiation Exposure
3
Sequencing RNA from OPTIMISTIC Trial
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood drawn during the OPTIMISTIC trial was collected in Tempus tubes and centrally stored at the New Castle MRC Centre for Rare & Neuromuscular Diseases biobank with strict SOPs and temperature control (−80°C). RNA was locally isolated in Nijmegen using the Tempus Spin RNA Isolation Kit (Applied Biosystems/Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration and RNA Integrity Number (RIN) were checked using Fragment Analyzer (Thermo Fisher Scientific). The mean RIN value was 8.9 and all were > 7.5. Hemoglobin mRNA was depleted using the Globinclear kit (Thermo Fisher Scientific). Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) according to the manufacturer’s instructions for a polyA mRNA workflow using UMI-indexed adapters. The size distribution (between 300 and 500 bp) was confirmed using Fragment Analyzer. A total of 150-bp paired end sequencing was performed with a NovaSeq6000 machine (Illumina) at a library concentration of 1.1 nM, generating > 30 M read pairs per sample. All raw sequencing data and associated genotype/phenotype/experimental information is stored in the European Genome-phenome Archive (EGA) under controlled access with Dataset ID EGAS00001005830 [28 ].
4
Purification and Transcriptome Analysis of Human RBCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transcriptome analysis was performed as previously described.14 (link) In short, to purify human RBCs we followed a method originally developed by Beutler et al.15 (link) To evaluate the purification of the RBCs, we used the gelatin zymography technique, previously described by Achilli et al.16 (link) This method allows the detection of contaminations with polymorphonuclear neutrophils, a type of leucocyte that cannot be eliminated by washing the blood sample. Polymorphonuclear neutrophils are the only type of blood cells that express the matrix metalloproteinase 9, whose catalytic activity against gelatin can be used as a specific marker.
Fluorescence-activated cell sorting was performed with wild-type (WT) murine blood samples using a FACSAria III (Becton Dickinson, Franklin Lakes, NJ).
For RNA isolation, we used the RiboPure RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA) and 500 μL of human or murine blood samples, prepared as described before. Subsequently, the α- and β-globin messenger RNAs, which have the highest expression in reticulocytes, were removed from the total RNA preparations by using the GLOBINclear Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Transcriptome analysis was performed by Expression Analysis Inc (Durham, NC) using next-generation sequencing. More information are detailed in thesupplemental Material .
Fluorescence-activated cell sorting was performed with wild-type (WT) murine blood samples using a FACSAria III (Becton Dickinson, Franklin Lakes, NJ).
For RNA isolation, we used the RiboPure RNA Purification Kit (Thermo Fisher Scientific, Waltham, MA) and 500 μL of human or murine blood samples, prepared as described before. Subsequently, the α- and β-globin messenger RNAs, which have the highest expression in reticulocytes, were removed from the total RNA preparations by using the GLOBINclear Kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. Transcriptome analysis was performed by Expression Analysis Inc (Durham, NC) using next-generation sequencing. More information are detailed in the
5
Transcriptomic Analysis of Innate Immunity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples for transcriptomic analysis of the innate immune response were collected in Paxgene®; tubes at weeks 0 (days 0 and 1), 7 and 8 (days 56 and 57). RNA was isolated using the PAXgene Blood RNA kit (Qiagen) according to the manufacturer's protocol. Following globin depletion (GLOBINclear™ Kit, ThermoFisher SCIENTIFIC) total RNA was quantified using a NanoDrop spectrophotometer. Transcription amplification and labeling (Illumina®; TotalPrep™-96 RNA Amplification Kit, Ambion) was performed starting with 50 ng RNA per sample. After hybridization (Whole-Genome Gene Expression Direct Hybridization, Illumina), the slides were scanned on the Illumina iScan system (Wellcome Trust Centre for Human Genetics, University of Oxford).
6
RNA Extraction and Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TRIzol samples were thawed, mixed rigorously with 0.2 volumes of cold chloroform,and incubated for 3 min at RT. After centrifugation for 30 min at 4°C and the maximum speed, the supernatant was carefully transferred to a new tube and mixed with an equal volume of 70% ethanol. Subsequently, the manufacturer’s instructions from the RNeasy MinElute Kit (Qiagen) were followed with DNase digestion (DNase I, Qiagen) for 30 min on column. Elution of the RNA was carried out in 14 µl. Human globin mRNA was depleted from all samples except from samples #1 and #2 using the GLOBINclear kit (ThermoFisher Scientific). The quality of the RNA was assessed using the Agilent 6000 Pico kit with Bioanalyzer 2100 (Agilent), and the RNA quantity was assessed using the Qubit RNA HA assay kit and a Qubit 3.0 fluorometer (ThermoFisher Scientific). Upon arrival at BGI Genomics Co (Hong Kong), the RNA quality of each sample was double-checked before sequencing. The median RIN value over all ex vivo samples was 6.75 (IQR: 5.93–7.40) (Figure 2—figure supplement 4 ), although this measurement has only limited significance for samples containing RNA of two species. Customized library construction in accordance with Tonkin-Hill et al., 2018 (link), including amplification with KAPA polymerase and HiSeq 2500 100 bp paired-end sequencing, was also performed by BGI Genomics Co (Hong Kong).
7
Whole Blood RNA-Seq Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from whole blood using PAXgene Blood RNA Kit (PreAnalytiX), followed by treatment with GLOBINclear™ kit (ThermoFisher Scientific) to remove unwanted globin mRNA. The remaining RNA product was used to prepare cDNA library using Illumina TruSeq Stranded mRNA library preparation kit (Illumina). RNAseq was performed by Beijing Genomics Institute using the Illumina Hiseq2000 with 75 base-pair (bp) paired-end reads to a depth of at least 30 million reads per sample.
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
RNAseq output was processed as previously described [76 (link)]. Read pairs were pre-processed to adjust base calls with phred <5 to ‘N’ and to remove read pairs where either end had fewer than 30 unambiguous base calls. This method indirectly removes read pairs containing mostly adaptor sequences. Read pairs were aligned to the human genome (hg19) using STAR (v2.3.1d) [77 (link)]. Gene counts were tabulated using htseq (v0.6.0) with the intersection-strict setting turned on and Ensembl gene annotations (GRCh37.74) used to map genomic locations to gene identifiers [78 (link)]. The edgeR (v3.36.0) package function, cpm, was used to calculate TMM-normalized counts-per-million (CPM) expression matrices [79 (link)].
8
Microarray Analysis of Gene Expression from Whole Blood
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from the PAXgene tubes using the PAXgene Blood miRNA Kit (Qiagen), and from the QFT-TB Plus stimulated samples, which had been lysed in RNAprotect, using the RNEasy mini kit (Qiagen), according to the manufacturer's instructions, incorporating on-column DNAse digestion. Globin depletion was performed using the GLOBINclear Kit (ThermoFisher), RNA was quantified by Nanodrop and the quality was assessed using an Agilent Bioanalyzer (Agilent, Cheshire, UK. The two-color low input Quick Amp Labelling kit (Agilent) was used to Cy3-or Cy5-fluorescently label cRNA samples, which were then hybridized to SurePrint G3 Human Gene Expression 60K GeneChip microarrays (Agilent) according to the manufacturer's instructions. Hybridization intensity was quantified via a SureScan Microarray Scanner (Agilent). Microarray data are deposited at Gene Expression Omnibus, Series GSE153342 .
Individual channel intensities from the GeneChip data were extracted independently and analysed as separate observations [4 ].
Individual channel intensities from the GeneChip data were extracted independently and analysed as separate observations [4 ].
9
Transcriptome and Genome Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA sequencing data was generated from the same stock of total RNA samples used for miRNA profiling. For each sample, 1 µg of total RNA was subject to globin-mRNA depletion using the GlobinClear kit (Thermo Fisher) followed by library preparation using 500 ng of high-quality globin-depleted RNA (RIN > 8) and KAPA Stranded RNA-Seq kit with RiboErase (Kapa Biosystems). The libraries were quality-checked and quantified prior to multiplexing and 100 bp paired-end sequencing using the TruSeq SBS Kit v3 - HS (Illumina) on a HiSeq 2500 instrument (Illumina). Genomic DNA libraries were constructed using the TruSeq Nano DNA Library Prep Kit (Illumina) and sequenced at 30× coverage on the HiSeq X platform (Illumina) at the Australian Genome Research Facility in Melbourne, Australia.
10
RNA Sequencing and Gene Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gene expression analysis was performed, as previously described (29 (link)). Primers used to detect the specific genes are listed in Table S4 . For the RNA sequencing studies, total RNA was isolated from blood via cardiac puncture using a Blood RNA isolation kit (ThermoFisher Scientific) and subsequently, globin mRNA was depleted using GLOBINclear kit (ThermoFisher Scientific). RNA was sequenced on NextSeq500 system and the library was prepared with the Agilent SureSelect Stranded mRNA kit.
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!