Single cells are captured and barcoded using 10X Chromium platform (10X Genomics). scRNA-Seq libraries were prepared following the instructions from Chromium Single Cell 3′ Reagent Kits User Guide (v2 or v3). Briefly, Gel Bead-In EMulsions (GEMs) are generated using single cell preparations. After GEM-RT and cleanup, the cDNA from barcoded single cell RNAs were amplified before quantification using Agilent Bioanalyzer High Sensitivity DNA chips. The single cell 3′ gene expression libraries were constructed and cDNA corresponding to an insertion size of around 350 bp selected. Libraries were quantified using Agilent Bioanalyzer High Sensitivity DNA chip and pooled together to get similar numbers of reads from each single cell before sequencing on a NovaSeq S4 lane (Novogene). For three samples (patients 1–3), capture and NGS were performed by the Cedars-Sinai Applied Genomics, Computation and Translational Genomics Core; for the remaining samples capture and library preparation were performed in-house.
Agilent bioanalyzer dna high sensitivity chip
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Bioanalyzer DNA high sensitivity chip is a lab equipment product designed to analyze DNA samples. It provides accurate and sensitive quantification of DNA fragments and libraries.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer rna nano chip, by Agilent Technologies (6 mentions)
Hiseq 4000, by Illumina (4 mentions)
Hiseq 2500, by Illumina (3 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Agilent bioanalyzer, by Agilent Technologies (3 mentions)
Chromium platform, by 10x Genomics (3 mentions)
Truseq stranded mrna library preparation protocol, by Illumina (3 mentions)
Truseq stranded mrna sample preparation guide, by Illumina (3 mentions)
Truseq stranded total rna protocol, by Illumina (3 mentions)
Barcoded adapters, by Illumina (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Nextera xt index v2 kit a and b, by Illumina (2 mentions)
Nextseq 500 system, by Illumina (2 mentions)
Nextseq500 high output v2 flowcell, by Illumina (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Truseq small rna sample prep kit, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Truseq small rna sample preparation kit, by Illumina (2 mentions)
Thermocycler, by Bio-Rad (2 mentions)
52 protocols using agilent bioanalyzer dna high sensitivity chip
1
Single-cell RNA-seq protocol using 10X Chromium
2
Single-Cell Sequencing Using 10x Genomics
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Single-cell sequencing was performed using on the 10x Genomics Chromium technology according to the manufacturer’s protocol (Single Cell 3’ Reagent Kits v2, 10x Genomics, USA): Cell suspensions, reverse transcription master mix and partitioning oil were loaded on a single-cell “A” chip, then run on the Chromium Controller. mRNA was reversed transcribed to cDNA within the droplets at 53 °C for 45 min. cDNA was amplified for 12 cycles in total on a BioRad thermocycler. SpriSelect beads (Beckman Coulter, USA) were used for size selection of cDNA based on a ratio of SpriSelect reagent volume to sample volume of 0.6. For quality control, cDNA was analyzed on Agilent Bioanalyzer high-sensitivity DNA chips. cDNA was enzymatically fragmented for 5 min at 32 °C, followed by end repair and A-tailing at 65 °C for 30 min. A double-sided size selection of the cDNA was performed using SpriSelect beads. After adapter ligation, cDNA was amplified using a sample-specific index oligo as primer, followed by a last double-sided size selection using SpriSelect beads. Final cDNA libraries were analyzed again on an Agilent Bioanalyzer high-sensitivity DNA chip. Libraries were sequenced on a HiSeq 4000 Illumina platform aiming for 150 million reads per library.
3
Single-cell RNA-seq using 10X Chromium
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Single cells were captured and barcoded using the 10X Chromium platform (10X Genomics). scRNA-seq libraries were prepared following the instructions from the Chromium Single Cell 3ʹ Reagent Kits User Guide (v3). Briefly, Gel Bead-In EMulsions (GEMs) were generated using single-cell preparations. After GEM-RT and cleanup, the complementary DNAs (cDNAs) from barcoded single-cell RNAs were amplified before quantification using Agilent Bioanalyzer High Sensitivity DNA chips. The single-cell 3′ gene expression libraries were constructed and cDNA corresponding to an insertion size of ∼ 350-400 bp were selected. Libraries were quantified using Agilent Bioanalyzer High Sensitivity DNA chips and pooled together to get similar numbers of reads from each single cell before sequencing on the NovaSeq S4 (Novogene).
4
Illumina Library Preparation and Sequencing
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cDNA resulting from PCR reactions described above was quantified using the Picogreen Assay (Invitrogen), and the sizes of products were confirmed using the Agilent Bioanalyzer DNA high Sensitivity chip (Agilent). A 50 µL index PCR reaction was carried out to attach dual indices and Illumina sequencing adapters. Twenty-five microliters of 2× KAPA HiFi HotStart Ready mix was combined with 5 µL Nextera XT Index primer 1 (N7xx) and Index primer 2 (S5xx) and added to 2 ng of cDNA for the PCR reaction. AMPure XP beads (Beckman Coulter Genomics) were used to purify the final libraries. Libraries were then quantified using the Picogreen Assay and Library Quantification kit (Kapa Biosystems). Agilent Bioanalyzer DNA high Sensitivity chip (Agilent) was used to confirm the sizes of the cDNA libraries. The libraries were normalized and pooled. The pooled libraries were then sequenced using Illumina MiSeq 300 cycle paired-end sequencing.
5
10X Genomics Single-Cell RNA-Seq Protocol
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For 10X Genomics single-cell RNA sequencing, cells were brought to a concentration of about 1000 cells/µL using the resuspension buffer. A total of 8000 cells were targeted for cDNA preparation and put through the 10X Genomics Chromium Next GEM Single Cell 3′ v3.1 (Pleasanton, CA, USA) pipeline for library preparation. After cDNA preparation and GEM generation and clean-up, cDNA was quality checked and quantified on an Agilent Bioanalyzer DNA High-Sensitivity chip at a 1:10 dilution. Successful traces showed a fragment size range of approximately 1000–2000 bp. A total of 12 indexing cycles for the library were used. The quality of the indexed libraries was checked on an Agilent Bioanalyzer DNA High-Sensitivity chip at a 1:10 dilution. Successful libraries had fragment size distributions with peaks around 400bp. Libraries were sequenced using 4 Illumina Hiseq lanes (Paired-end 75 bp).
6
RNA Extraction and Sequencing from Brain Tissue
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The brain tissues were dissected rapidly, flash-frozen in liquid nitrogen, and maintained on dry ice until storage at –80°. Frozen brain tissue was homogenized in 1 ml TRIzol solution (Life Technologies 15596-018), using a motorized homogenizer. Samples were then centrifuged at 4° for 10 min at 4000 rpm to separate out debris, and the resulting supernatant was processed for total RNA extraction. Total RNA was treated with TURBO DNase (Ambion AM223) and purified with Zymo Research RNA Clean & Concentrator-25kit (Zymo Research R1018). The DNase treated and purified total RNA (5 μg for cerebellum, neocortex, and hippocampus, or 1 μg for hypothalamus) was used for subsequent mRNA isolation and RNA-seq library preparation. The External RNA Controls Consortium (ERCC) RNA Spike-in Controls (Ambion 4456740) were diluted 1:10 (for 5 μg total RNA sample) and 1:100 (for 1 μg total RNA sample) and added to total RNA. RNA-seq libraries were generated using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs Inc. E7530L), with 12 PCR cycles. The qualities of the libraries were examined using Agilent High Sensitivity DNA Bioanalyzer Chips (Agilent Technologies 5067-4626) and quantified by KAPA Library Quantification Kits for Illumina sequencing platforms (KAPA Biosystems KK4824).
7
16S Bacterial DNA V4 Region Sequencing
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The 16S bacterial DNA V4 region was amplified from the extracted DNA by PCR and sequenced in the Illumina MiSeq Reagent Kit v2 flowcell on an Illumina MiSeq System using 2 150bp paired-end protocol. All samples were sequenced using uniquely barcoded primers (515FB: 5’-GTG YCA GCM GCC GCG GTA A-3’; 806RB: 5’-GGA CTA CNV GGG TWT CTA AT-3’)47 (link) that were modified from the original 515F-806R primer pairs9 (link). Agilent High Sensitivity DNA Bioanalyzer chips was used to assess the library qualities. The number of reads in each sample ranged from 22 thousand to 1 million. The mean number of reads for the samples was 145 thousand with standard deviation of 235 thousand. The histograms of the numbers of reads in the samples are given in supplementary Fig. S1 (A ).
8
Illumina-Based Metagenomic Library Preparation
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Metagenomic libraries were constructed using the Nextera XT DNA Library Preparation Kit (Illumina) and Illumina Nextera XT Index v2 Kit A and B following the manufacturer’s protocols. Library qualities were assessed on Agilent High Sensitivity DNA Bioanalyzer chips. Libraries were pooled and sequenced on an Illumina NextSeq500 High Output v2 flowcell on an Illumina NextSeq 500 System, producing 2 150bp paired-end reads. All samples and control reads were pre-processed, and quality filtered using Trim_Galore . Host-derived reads were removed using KneadData . The number of reads in each sample ranged from 400 thousand to 7,258 thousand. The mean number of reads of the samples was 3,465 thousand with standard deviation of 1,829 thousand. The histograms of the numbers of reads in the samples are given in supplementary Fig. S1 (B ).
9
Metagenomic Sequencing Library Preparation
10
Illumina TruSeq DNA Exome Enrichment
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DNA library preparation was completed using Illumina's TruSeq DNA Sample Prep v2 kit protocol (Illumina, CA). The DNA (1 μg) was randomly fragmented by the Covaris S220 (Covaris, MA) using insert sizes of 100 to 900 bp. DNA quality was checked by analysis of samples on the Agilent 2100 Bioanalyzer using a DNA High Sensitivity chip followed by quantification on the Qubit 2.0 Fluorometer (Life Technologies).
Exome enrichment was carried out using the standard protocol for the Illumina TruSeq Exome Enrichment Kit (Illumina, CA). Combining 500 ng from each sample creates library pools (only samples with different index adapters were pooled together). Following the Illumina Trueseq Exome Enrichment protocol, the quality of the final libraries is checked using an Agilent High Sensitivity DNA Bioanalyzer chip (Agilent, CA). The libraries are then quantified using the Qubit 2.0 Fluorometer and normalized to 1 ng/ul.
Exome enrichment was carried out using the standard protocol for the Illumina TruSeq Exome Enrichment Kit (Illumina, CA). Combining 500 ng from each sample creates library pools (only samples with different index adapters were pooled together). Following the Illumina Trueseq Exome Enrichment protocol, the quality of the final libraries is checked using an Agilent High Sensitivity DNA Bioanalyzer chip (Agilent, CA). The libraries are then quantified using the Qubit 2.0 Fluorometer and normalized to 1 ng/ul.
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