RNA was isolated from f-hAFS-EVs and p-hAFS-EVs with miRNeasy Micro Kit (Qiagen, Milan, Italy) according to the manufacturer’s instructions. RNA integrity and size distribution were evaluated using the Agilent Small RNA Kit with the small noncoding RNA chip in order to assess the content of small RNAs ranging from 6 to 150 nucleotides (nt). The Qubit microRNA Assay Kit (Thermo Fisher Scientific, Monza, Italy) was used to quantify microRNAs (miRNAs) content, following the manufacturer’s instructions. miRNA sequencing libraries were prepared and amplified using QIAseq miRNA Library kit (Qiagen, Milan, Italy) using 18.5 ng of isolated miRNAs as input and following the manufacturer’s instructions. Libraries were pooled after quality check and quantification by TapeStation (Agilent Technologies, Foster City, CA, USA) was performed using Agilent High Sensitivity D1000 ScreenTape. Pooled libraries were assessed for quality control by real-time qPCR following “Sequencing Library qPCR Quantification” Guide (Illumina Inc., San Diego, CA, USA) and sequenced by Illumina NextSeq platform using High Hutput Hit v2.5 (75 cycles) (Illumina Inc., San Diego, CA, USA). Base calling was performed with default Illumina NextSeq500 workflow.
Agilent high sensitivity d1000 screentape
Manufactured by Agilent Technologies
Sourced in United States
The Agilent High Sensitivity D1000 ScreenTape is a laboratory instrument designed for the analysis of DNA samples. It provides rapid and accurate quantification and sizing of DNA fragments in the range of 35 to 1,000 base pairs.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (5 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Stranded mrna prep, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Miseq v2, by Illumina (2 mentions)
Superscript 4 reverse transcriptase enzyme, by Thermo Fisher Scientific (2 mentions)
Nextera xt library preparation kit, by Illumina (2 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Ribo zero gold, by Illumina (2 mentions)
Picogreen assay, by Thermo Fisher Scientific (2 mentions)
Truseq stranded total rna lt sample preparation kit, by Illumina (2 mentions)
Truseq stranded total rna sample preparation guide, by Illumina (2 mentions)
Agilent 4200 tapestation, by Agilent Technologies (2 mentions)
Hiseq 4000 sequencer, by Illumina (2 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Qubit microrna assay kit, by Thermo Fisher Scientific (1 mentions)
Nextseq platform, by Illumina (1 mentions)
Mirneasy micro kit, by Qiagen (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
11 protocols using agilent high sensitivity d1000 screentape
1
Profiling of microRNA Content in extracellular vesicles
2
RNA Extraction from Toxoplasma Stages
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Total RNA was extracted from the purified tachyzoites and bradyzoites of the ME49ΔKu80Δhxgprt AtTIR1 strain and derivatives with TRIzol according to the manufacturer’s instructions (Invitrogen). The RNA integrity was verified using Agilent High Sensitivity D1000 ScreenTape in an Agilent 2200 TapeStation System (Agilent Technologies). Two biological replicates were analyzed.
3
Optimized DNA Fragmentation and Library Prep
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DNA of the dilution series was fragmented by sonication using Covaris Sonalab 7.1 S220 [Covaris, Woburn, MA, USA] (80 s, peak power 140.0, duty factor 10.0, cycles/burst 200, power ~ 12, temp below 12 °C). Shearing results were checked by electrophoresis using an Agilent D1000 screen tape [Agilent, Santa Clara, CA, USA]. The mean size of the fragmented DNA was ~300 bp. Sample preparation was performed using the NEBNext Ultra II DNA Library Prep Kit for Illumina sequencing [New England Biolabs, Ipswich, MA, USA] using an input amount of 1 µg DNA in 50 µl Tris-EDTA. NEB adapters were substituted for unique molecular identifier (UMI) TruSeq dual-index duplex adapters (15 µM) [Integrated DNA Technologies (IDT), Coralville, IA, USA], and USER enzyme steps were skipped. UMIs are used to remove duplicate reads and reduce the error rate during the data-analysis procedure. A size-selection to 300–400 bp using AMPure XP Beads [Beckman Coulter, Indianapolis, IN, USA] was performed after adding adapters. IDT xGen Library Amplification primers (5 µM p5 and 5 µM p7) were used to enrich the adapter-ligated DNA using PCR (12 cycles). The amplified product was measured by electrophoresis using the Agilent High Sensitivity D1000 screen tape after cleanup with AMPure XP Beads.
4
Transcriptomics of Chemo-Sensitive and Chemo-Resistant PDOs
5
Comprehensive RNA and cDNA Quantification
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RNA: The Qubit 4. fluorometer and the Qubit Assay Kits (Invitrogen) were used for the measurement of RNA concentration. The RNA BR Assay was applied for the quantitation of total RNA samples whereas the RNA HS Assay was utilized for the poly(A) + and ribodepleted RNA fractions. The quality of the total RNA samples was checked by using the Agilent TapeStation 4150 device, RNA ScreenTape and reagents were used. RIN scores above 9.6 were used for cDNA production. The RNA quality was assessed with the Agilent 2100 Bioanalyzer (for PacBio sequencing) or Agilent 4150 TapeStation System (for MinION sequencing) and RIN scores above 9.6 were used for cDNA production.
cDNA: The Qubit dsDNA HS Assay Kit (Invitrogen) was used to quantify the cDNA samples. For the analysis of Illumina library quality, the Agilent High Sensitivity D1000 ScreenTape was used.
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6
mRNA Isolation and Sequencing of CD8+ T Cells
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mRNA‐isolation from a minimum of 1 × 105 cells of the CD8+ T cell subsets was performed immediately after cell sorting using the RNeasy Micro Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentrations and the RNA integrity number (RIN) were determined with the Agilent 4150 Tape Station instrument using the Agilent High‐Sensitivity RNA Screen Tape (Agilent, Santa Clara, CA). All samples that were used for sequencing had RINe values greater than or equal to 8.8. Libraries were prepared with 14 ng RNA input per sample using the QuantSeq 3′mRNA‐Seq Library Prep Kit‐FWD and the UDI 12 nt Unique Dual Indexing Add‐on kit (both Lexogen, Vienna, Austria) according to the manufacturer's instructions. For library amplification the optimal number of cycles was assessed for each sample. The quality and concentration of the libraries was determined on an Agilent 4150 Tapestation instrument using the Agilent High Sensitivity D1000 ScreenTape (Agilent, Santa Clara, CA). The pooled cDNA libraries (libraries were diluted to 2 nM concentration) were sequenced in an Illumina NextSeq 2000 instrument (Illumina, San Diego, CA). These sequence data have been submitted to the GEO database under accession number GSE207621. The QuantSeq data analysis pipeline (Lexogen, Vienna, Austria) was used for data processing and the calculation of normalised counts.
7
Viral RNA Sequencing from Deer Mice
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Viral RNA from positive deer mouse samples was prepared for Next-generation sequencing. Briefly, cDNA was generated using SuperScript IV Reverse Transcriptase enzyme (Invitrogen) with random hexamers. PCR amplification was performed using ARTIC network V3 tiled amplicon primers in two separate reactions by Q5 High-fidelity polymerase (NEB). First-round PCR products were purified using Ampure XP beads (Beckman Coulter). Libraries were prepared using the Nextera XT Library Preparation Kit (Illumina) according to manufacturer protocol. Unique Nextera XT i7 and i5 indexes for each sample were incorporated for dual indexed libraries. Indexed libraries were again purified using Ampure XP bead (Beckman Coulter). Final libraries were pooled and analyzed for size distribution using the Agilent High Sensitivity D1000 Screen Tape on the Agilent Tapestation 2200, final quantification was performed using the NEBNext Library Quant Kit for Illumina (NEB) according to manufacture protocol. Libraries were then sequenced on the Illumina MiSeq V2 using 2 x 250 paired end reads.
8
SARS-CoV-2 Sequencing from Deer Mice
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Viral RNA from positive deer mouse samples was prepared for Next-generation sequencing. Briefly, cDNA was generated using SuperScript IV Reverse Transcriptase enzyme (Invitrogen) with random hexamers. PCR amplification was performed using ARTIC network V3 tiled amplicon primers in two separate reactions by Q5 High-fidelity polymerase (NEB). First-round PCR products were purified using Ampure XP beads (Beckman Coulter). Libraries were prepared using the Nextera XT Library Preparation Kit (Illumina) according to manufacturer protocol. Unique Nextera XT i7 and i5 indexes for each sample were incorporated for dual indexed libraries. Indexed libraries were again purified using Ampure XP bead (Beckman Coulter). Final libraries were pooled and analyzed for size distribution using the Agilent High Sensitivity D1000 Screen Tape on the Agilent Tapestation 2200, final quantification was performed using the NEBNext Library Quant Kit for Illumina (NEB) according to manufacture protocol. Libraries were then sequenced on the Illumina MiSeq V2 using 2 × 250 paired end reads.
9
Transcriptomics of Chemo-Sensitive and Chemo-Resistant PDOs
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Total RNA was isolated using the RNeasy Mini Kit (QIAGEN). RNA concentration and purity were assessed by a NanoDrop Spectrophotometer. RNA integrity number (RIN) was assessed with the TapeStation. Libraries were prepared from 1 μg of RNA using Illumina Stranded mRNA Prep (Illumina). Library concentration and quality were measured using the Qubit dsDNA HS Assay kit (Life Technologies) and Agilent High Sensitivity D1000 ScreenTape (Agilent Technologies). RNA sequencing was performed by Illumina NovaSeq 6000 platform for paired-end sequencing, 150-bp read length, for ∼20 million raw reads per sample. Data analysis according to RNA sequencing FASTQ files was processed with HISAT2 Aligner in default mode with the Ensembl human transcriptome annotation (Build version GRCh38 and transcript annotation GRCh38.89). Differential gene expression analysis was performed using DESeq2 software (version 1.34.0), using an expression analysis cutoff above 1 Fragment Per Kilobase of transcript per Million mapped reads (FPKM). The fold transcript level changes (represented by FPKM) were calculated from of a list of 78 genes related to the apoptosis process (GO: 0006915) comparing paclitaxel- and vehicle-treated PDOs. The fold induction of the genes in chemo-sensitive HCI-002 versus chemo-resistant HCI-001 PDOs was calculated to establish the rank list (Figure 1c ).
10
Illumina TruSeq Stranded Total RNA Sequencing
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RNA-seq libraries were generated as described in TruSeq Stranded Total RNA Sample Preparation Guide (illumina, part no. 15031048 Rev. E October 2013) using Illumina TruSeq Stranded Total RNA LT sample preparation kit. Ribosomal depletion step was performed on 500 ng of total RNA using Ribo-Zero Gold (Illumina, 20020598 and 20020492) followed by a 8 min heat fragmentation step aimed at producing libraries with an insert size between 120bp-200bp. First strand cDNA was synthesized from the enriched and fragmented RNA using SuperScript II Reverse Transcriptase (Thermofisher, 18064014) and random primers. Second strand synthesis was performed in the presence of dUTP. Following 3′ adenylation and ligation of adaptors to the dsDNA, libraries were subjected to 13 cycles of PCR. RNA-seq libraries were quantified using PicoGreen assay (Thermofisher, P11496) and sized and qualified using an Agilent 4200 TapeStation with Agilent D1000/High sensitivity ScreenTape (Agilent, 5067–5584). Libraries were normalized to 4nM and pooled before clustering using a cBot2 followed by 75bp paired-end sequencing on a HiSeq 4000 sequencer (illumina). PDCL normalized RNA expression data is provided in Table S1 .
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