Neurons, astrocytes, and microglia from the cerebral MPSs were jointly collected, and mRNA was extracted using the PureLink RNA mini kit (Thermo Fisher Scientific, catalog no. 12183018A). Total RNA was analyzed and quantified using the Fragment Analyzer (Advanced Analytical). RNA sequencing (RNA-seq) libraries were prepared using a volume reduced version of the New England Biolabs ribosomal reduction chemistry and RNA-seq library construction kit (Mildrum et al., in preparation). In brief, RNA quality and quantity were confirmed using an Agilent Fragment Analyzer and ribosomal RNA (rRNA) was depleted from 50 ng of total RNA using the NEBNext Ribodepletion kit (New England Biolabs) at a 1:6 ratio from the standard protocol using a Mosquito HV automated liquid handler (TTP Labtech). The resulting depleted RNA is then fragmented and converted to cDNA, and indexed Illumina libraries are constructed using the NEBNext Ultra II Directional RNA Library Construction Kit (New England Biolabs) at a 1:10 ratio from the standard protocol using the Mosquito HV. Final libraries are quantified using SYBRgreen on a Varioskan plate reader and spot-checked using the Agilent Fragment Analyzer. Samples were pooled and quantified by quantitative polymerase chain reaction before Illumina sequencing on a HiSeq2000 using 40-nt single-end reads.
Fragment analyzer
Manufactured by Agilent Technologies
Sourced in United States, Germany, France, Canada, Italy
The Fragment Analyzer is a capillary electrophoresis-based instrument designed to separate and analyze DNA, RNA, and protein fragments. It provides rapid and accurate size determination and quantification of fragments ranging from 50 to 40,000 base pairs.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (63 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (61 mentions)
Nanodrop, by Thermo Fisher Scientific (53 mentions)
Novaseq 6000, by Illumina (49 mentions)
Rneasy mini kit, by Qiagen (49 mentions)
Trizol reagent, by Thermo Fisher Scientific (45 mentions)
Hiseq 2500, by Illumina (41 mentions)
Trizol, by Thermo Fisher Scientific (39 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (34 mentions)
Ampure xp bead, by Beckman Coulter (32 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (32 mentions)
Hiseq 4000, by Illumina (31 mentions)
Bioanalyzer, by Agilent Technologies (18 mentions)
Rneasy plus mini kit, by Qiagen (18 mentions)
Kapa library quantification kit, by Roche (18 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (17 mentions)
Rneasy kit, by Qiagen (16 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (16 mentions)
Miseq platform, by Illumina (15 mentions)
Qiazol lysis reagent, by Qiagen (13 mentions)
884 protocols using fragment analyzer
1
Cerebral Multi-cell Type Transcriptomics
2
Transcriptomic Analysis of Primary Brain Endothelial Cells
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Ch25hfl/fl and Ch25hECKO female mice were injected with tamoxifen. Nine brains per genotype were pooled and primary brain microvascular endothelial cells (pMBMEC) were isolated and plated in a 96‐well plate (2 wells/brain). Confluent pMBMEC were left unstimulated or stimulated IL‐1β for 24 h. RNA of three wells was pooled to obtain one replicate for RNA sequencing. The Lausanne Genomic Technologies Facility performed the RNA‐seq. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies), and all RNAs had a RQN between 8.7 and 10. RNA‐seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).
Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina).
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Ch25hfl/fl and Ch25hECKO female mice were injected with tamoxifen. Nine brains per genotype were pooled and primary brain microvascular endothelial cells (pMBMEC) were isolated and plated in a 96‐well plate (2 wells/brain). Confluent pMBMEC were left unstimulated or stimulated IL‐1β for 24 h. RNA of three wells was pooled to obtain one replicate for RNA sequencing. The Lausanne Genomic Technologies Facility performed the RNA‐seq. RNA quality was assessed on a Fragment Analyzer (Agilent Technologies), and all RNAs had a RQN between 8.7 and 10. RNA‐seq libraries were prepared from 500 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy, and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies).
Cluster generation was performed with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents and sequenced on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single end). Sequencing data were demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina).
3
RNA-sequencing protocol for rice
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Quality of RNA was checked by determining the RNA Integrity Number (RIN) with a Fragment Analyzer (Agilent). For the library preparation, samples with a RIN value > 6 were used. Eighteen RNA libraries were prepared using an Illumina TruSeq stranded mRNA sample preparation kit by MGX-Montpellier GenomiX core facility (MGX) France (https://www.mgx.cnrs.fr/ ). Library construction and sequencing were performed as described in Karmakar et al. (2019) (link) on an Illumina HiSeq 2500. The quantitative and qualitative validation of the library was performed by qPCR, Roche LightCycler 480, and a Fragment Analyzer (Agilent) using a Standard Sensitivity NGS kit. Quality control and assessment of raw Illumina reads in FASTQ format were done by FastQC software (version 0.11.5) to obtain per base quality, Guanine-Cytosine (GC) content, and sequence length distribution. Clean reads were obtained by removing the low-quality reads, adapters, and poly-N-containing reads by using Trimmomatic v0.36 software (Bolger et al., 2014 (link)). RNAseq reads were aligned to the IRGSP 1.0 version of the rice genome using HISAT2 v2.0.5.1 (Kim et al., 2015 (link)). The number of reads mapped to each gene locus was counted using HTSEq-count v0.6.0 (Anders et al., 2015 (link)).
4
RNA Extraction and RNA-Seq Analysis in Mice Kidneys
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RNA from frozen half-kidneys of 72 mice were extracted and purified using RNAeasy MiniElute Spin Column (Qiagen). RNA quality was assessed on a Fragment Analyzer (Agilent Technologies). All RNAs had an RNA quality number (RQN) between 7.5 and 9.7. RNA-Seq libraries were prepared from 200 ng of total RNA with the Illumina TruSeq Stranded mRNA reagents (Illumina) using a unique dual indexing strategy and following the official protocol automated on a Sciclone liquid handling robot (PerkinElmer). Libraries were quantified by a fluorometric method (QubIT, Life Technologies) and their quality assessed on a Fragment Analyzer (Agilent Technologies). Clusters were generated with 2 nM of an equimolar pool from the resulting libraries using the Illumina HiSeq 3000/4000 SR Cluster Kit reagents. Sequencing was performed on the Illumina HiSeq 4000 using HiSeq 3000/4000 SBS Kit reagents for 150 cycles (single read). Sequencing data were demultiplexed, filtered for failed reads, and written to FASTQ files using the bcl2fastq2 conversion software (version 2.20, Illumina). Details for RNA-Seq reads mapping are described in Supplemental Methods .
5
RNA-Sequencing of Blood Samples
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RNA was isolated from peripheral blood buffy coat samples on the automated Chemagic 360 (Perkin Elmer) instrument according to the manufacturer’s instructions. Extracted RNA was quantitated by Qubit™ RNA BR Assay Kit (Thermo Fisher Scientific) followed by the RNA quality check using Fragment Analyzer (Agilent). For each sample, RNA libraries were prepared from 100ng RNA using the KAPA RNA HyperPrep Kit with RiboErase (Kapa Biosystems) according to manufacturer’s protocol, followed by quality check using Fragment Analyzer (Agilent) and quantification by qPCR (Kapa qPCR quantification kit, Kapa biosystem) on the LightCycler 480 (Roche). The libraries were normalized and pooled, and then sequenced using NovaSeq6000 platform (Illumina) to an average of 40M 101bp paired end reads per sample. Low-quality reads and adapter sequences were trimmed from the raw sequencing data with Trimmomatic [32 (link)]. The remaining reads were then aligned to human reference genome hg38 with STAR aligner [33 (link)]. Gene counts were quantified with the STAR --quantMode GeneCounts function. Fragments per kilobase of transcript per million mapped fragments (FPKM) were quantified with Cufflinks [34 (link)].
6
Temporal Dynamics of Gene Expression
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Cells were collected at 0 h, 24 h and 48 h post-induction as bulk samples, and on days 3, 4, and 6, cells were sorted into Venus-positive and Venus-negative cells using FACSAria III (BD bioscience). Cells were washed with PBS and processed using the Qiagen RNeasy mini kit. RNA was additionally treated with a TURBO DNA-free™ kit (Invitrogen) according to the manufacturer’s protocols. The integrity of RNA was measured using Fragment Analyzer™ (Advanced Analytical). RNAseq-Libraries were prepared using QuantSeq 3’ mRNA-Seq Library Prep Kit (Lexogen GmbH). LUTHOR 3’-scRNAseq (Lexogen GmbH) libraries were prepared using the manufacturer’s protocol after sorting cells into 5 μl lysis agent. Concentrations and distributions of the libraries were checked with the Fragment Analyzer™ using HS NGS Fragment Kit (Agilent; DNF-474-0500). Libraries were then pooled and sequenced using Illumina Nextseq550 in a single read 75 cycles run.
7
RNA-Seq Analysis of DETA/NO-Treated Cancer Cells
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Total RNA was extracted from DETA/NO-treated cancer cell lines using an RNeasy kit (Qiagen) according to the manufacturer’s instructions. After RNA quantification using a NanoDrop (Thermo Fisher Scientific) samples were sent for parallel sequencing to the Collaborative Health Initiative Research Program (CHIRP) based at Uniformed Services University (USU). Total RNA integrity was evaluated by automated capillary electrophoresis on a Fragment Analyzer (Agilent Technologies, Santa Clara, CA., USA). Sequencing libraries were prepared using the TruSeq Stranded mRNA Library Preparation Kit (Illumina, San Diego, CA, USA) with barcoded adapters, and the yield and concentration were determined using the Illumina/Universal Library Quantification Kit (KAPA) on the Light Cycler 480 (Roche, Indianapolis, IN., USA). Library size distribution was established by using the Fragment AnalyzerTM (Agilent Technologies). Clustering was done on cBotTM 2 (Illumina), and sequencing was performed on the HiSeq 3000 (Illumina) with paired-end reads of 75 bp in length with an intended depth of 40 million reads per sample. The resulting RNA-Seq data were exported as FASTQ files for the subsequent data analysis pipeline.
8
High-Throughput Genomic DNA Extraction and Sequencing
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Genomic DNA from dried leaf samples was isolated using the CTAB-based extraction protocol [9 (link), 23 ]. DNA concentration (ng/μl), average fragment size (bp) and quality (GQN = 300 bp) were measured using Fragment Analyzer (Agilent, US).
DNA library preparation was performed through an initial enzymatic fragmentation to 200–450 bp using NEBnext® Ultra™ II FS DNA Library Prep Kit for Illumina® (New England Biolabs, US) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, US). Highly-degraded DNA samples were processed without starting fragmentation using NEBnext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, US). DNA library double-size selection (320–470 bp) was done using SPRIselect® (Beckman Coulter, US). The average fragment size (bp) and molarity (nM) of the final products were calculated using Fragment Analyzer (Agilent, US).
DNA libraries were sent to RAPiD Genomics (Florida, US) and Fasteris SA (Plan-les-Ouates, Switzerland) for paired-end sequencing at low genome coverage sequencing (0.1-10X, 2x150 bp), aiming at a minimum of 5 million reads per sample, using HiSeq® 3000, as well as HiSeq® 4000 and NovaSeq® 6000 (Illumina, US).
DNA library preparation was performed through an initial enzymatic fragmentation to 200–450 bp using NEBnext® Ultra™ II FS DNA Library Prep Kit for Illumina® (New England Biolabs, US) and NEBNext® Multiplex Oligos for Illumina® (New England Biolabs, US). Highly-degraded DNA samples were processed without starting fragmentation using NEBnext® Ultra™ II DNA Library Prep Kit for Illumina® (New England Biolabs, US). DNA library double-size selection (320–470 bp) was done using SPRIselect® (Beckman Coulter, US). The average fragment size (bp) and molarity (nM) of the final products were calculated using Fragment Analyzer (Agilent, US).
DNA libraries were sent to RAPiD Genomics (Florida, US) and Fasteris SA (Plan-les-Ouates, Switzerland) for paired-end sequencing at low genome coverage sequencing (0.1-10X, 2x150 bp), aiming at a minimum of 5 million reads per sample, using HiSeq® 3000, as well as HiSeq® 4000 and NovaSeq® 6000 (Illumina, US).
9
RNA-seq of Peripheral Blood Mononuclear Cells
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RNA was extracted from cells (2.5 × 105 PBMCs) homogenized in 200 μL of Buffer RLT (Qiagen) and then extracted using the Quick-RNA MagBead Kit (Zymo) with DNase digestion. RNA quality was quantitated using Qubit HS RNA assays and assessed using a Fragment Analyzer (Agilent). Library preps were performed using the SMART-Seq v4 Ultra Low Input RNA Kit (Takara Bio) to synthesize full-length cDNA from an input of 10ng of RNA. After a bead-based clean-up to purify the cDNA, the Nextera XT kit was used to create libraries through a process of tagmentation and fragment amplification and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on a Fragment Analyzer (Agilent), pooled at equimolar concentrations, and sequenced on an Illumina NovaSeq6000 (Emory) at 100 bp, paired-end read length targeting ∼25 million reads per sample. Repeated measures from a group of PBMC samples collected from healthy controls and repeated measures of a subset of IMPACC samples were used across library prep and sequencing batches to assess inter-site batch effects throughout the study. Universal Human References controls were included to assess intra-site batch variation.
10
RNA Isolation and RNA-seq Library Preparation
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The quality of the isolated RNA from cell lines was determined with a fragment analyzer (Agilent, Santa Clara, CA, USA). Only the samples with a 260 nm/280 nm ratio between 1.8 and 2.1 and a 28S/18S ratio between 1.5 and 2 were further processed. The TruSeq Stranded mRNA (Illumina, Inc., San Diego, CA, USA) was used in the succeeding steps. Briefly, the total RNA samples (100–1000 ng) were poly A-enriched and then reverse-transcribed into double-stranded cDNA. The cDNA samples were fragmented, end-repaired, and adenylated before the ligation of TruSeq adapters containing unique dual indices (UDI) for multiplexing. Fragments containing TruSeq adapters on both ends were selectively enriched with PCR. The quality and quantity of the enriched libraries were validated using the fragment analyzer (Agilent, Santa Clara, CA, USA). The product was a smear with an average fragment size of approximately 260 bp (base pair). The libraries were normalized to 10 nM in 10 mM of Tris-Cl, pH 8.5, with 0.1% Tween 20. Methods for the library preparation of microdissected FFPE samples were followed as previously described [63 (link)].
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