One µL of each library were pooled together, quantified by Kapa Library Quantification Kit (Roche cat. # 07960140001). Samples were diluted to 4 nM, denatured with 0.2 N NaOH, and the library pool was further diluted to 12.5 pM prior to loading on an Illumina Miseq Nano kit for cluster and sequencing to determine amount required for deep sequencing. Libraries were pool based on Miseq read counts, diluted to 1.25 nM, denatured with 0.2 N NaOH, neutralized with 400 mM Tris-HCL pH 8 and diluted to a final loading concentration of 250pM. Pooled libraries were loaded onto an Illumina Novaseq6000 sequencer for cluster generation and sequencing to achieve a minimum of 20 million reads per sample using 100 bp single read.
Miseq nano kit
Manufactured by Illumina
Sourced in United States
The MiSeq nano kit is a lab equipment product from Illumina. It is designed for targeted sequencing of DNA samples. The kit provides the necessary reagents and consumables to perform a sequencing run on the MiSeq system.
Automatically generated - may contain errors
Lab products found in correlation
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (8 mentions)
Novaseq 6000, by Illumina (6 mentions)
2100 bioanalyzer high sensitivity kit, by Agilent Technologies (4 mentions)
2100 bioanalyzer rna 6000 nano assay, by Agilent Technologies (3 mentions)
Quant it ribogreen rna assay, by Thermo Fisher Scientific (3 mentions)
Truseq stranded total rna library prep kit, by Illumina (3 mentions)
4200 tapestation high sensitivity rna screentape assay, by Agilent Technologies (3 mentions)
5300 fragment analyzer ngs fragment kit, by Agilent Technologies (3 mentions)
4200 tapestation d1000 screentape assay, by Agilent Technologies (3 mentions)
Kapa library quantification kit, by Roche (2 mentions)
Gentra puregene tissue kit, by Qiagen (2 mentions)
Le220 ultrasonicator, by Covaris (2 mentions)
Hyperprep library preparation kits, by Roche (2 mentions)
Novaseq 6000 sequencer, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nextera indexing kit, by Illumina (1 mentions)
Ampure xp magnetic beads, by Beckman Coulter (1 mentions)
Mirvana rna isolation kit, by Thermo Fisher Scientific (1 mentions)
Blood and cell culture dna mini kit, by Qiagen (1 mentions)
12 protocols using miseq nano kit
1
Illumina Miseq and Novaseq6000 Sequencing
2
T-cell DNA Isolation and Sequencing
3
RNA Isolation and Transcriptome Analysis
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The RNEasy kit (Qiagen) was used to isolate RNA from BMDM cells incubated 20 h in DMEM 1% DMSO ± 20 μM h18:0, and the TRIzol reagent (ThermoFisher) was used to isolate RNA from RAW 264.7 cells incubated 20 h in DMEM 1% DMSO ± 100 μM h18:0. RNA was quantified using the Quant-iT RiboGreen RNA assay (ThermoFisher) and quality checked by the 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or 4200 TapeStation High Sensitivity RNA ScreenTape assay (Agilent) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions (Illumina, PN 20020599). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent), 4200 TapeStation D1000 ScreenTape assay (Agilent), or 5300 Fragment Analyzer NGS fragment kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (ThermoFisher) or by low pass sequencing with a MiSeq nano kit (Illumina). Paired end 100 cycle sequencing was performed on a NovaSeq 6000 (Illumina).
4
Exon Amplification and Sequencing
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Exon-specific primers linked to Illumina sequencing adapters were designed to generate amplicons spanning potentially novel exons using cDNA from day 0 (see Additional file 6 ). Primers were used in both convergent and divergent orientation to enable amplification of both circular and linear isoforms. Illumina sequencing indexes were added to each amplicon using a Nextera indexing kit (Illumina), and amplicons were sequenced to a target depth of 1500-6000 reads (depending on the number and intensity of visible products) using a MiSeq Nano Kit (Illumina) all according to manufacturer’s recommendations.
5
Amplifying Chromosomal eGFP for Deep Sequencing
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U2OS genomic DNA was prepared for deep sequencing by first amplifying short regions of the chromosomally encoded egfp. The oligos contained the necessary adapters for analysis by the Illumina MiSeq 250PE Nanokit are prDC174 to prDC179. Once amplified, the DNA fragments were purified with AMPure XP magnetic beads (Beckman Coulter). A 1.8× bead purification was used followed by two 80% ethanol washes. After washing, the DNA was eluted in 20 μl of sterile, nuclease-free water. These amplified DNA fragments were then indexed using an 8-cycle PCR amplification. Nextera indices 501, 502, 503, 504, 707, 708, and 709 were used in this work and are encoded within prDC185, prDC186, prDC180, prDC181, prDC187, prDC188, and prDC189, respectively. Once amplified, the PCR products were purified with AMPure XP magnetic beads similar to the first cleanup. These samples were quantified on a NanoDrop 2000c spectrophotometer and by a Qubit Fluorometer. DNA then diluted to 15 nM and samples with separate indices were pooled to a total volume of 20 μl. Samples were run on the 250-bp paired-end read Illumina MiSeq Nano kit. The sequencing results for each sample were then analyzed with CRISPResso using the standard parameters to evaluate indel formation (https://github.com/pinellolab/crispresso ).
6
RNA Sequencing of Brain Tissue and Organoids
7
DNA Extraction and NGS Library Preparation
8
T-cell DNA Isolation and Sequencing
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Genomic DNA from CD4+/CD8+ T-cells was isolated using a Gentra PureGene Tissue Kit (Qiagen) and sheared on a LE220 ultrasonicator (Covaris). Libraries were prepared from sheared DNA with HyperPrep Library Preparation Kits (Roche) and quantified using the Quant-iT PicoGreen dsDNA assay (Life Technologies) or low pass sequencing with a MiSeq nano kit (Illumina). Paired end 150 cycle sequencing was performed on a NovaSeq 6000 (Illumina).
9
Whole Genome Bisulfite Sequencing Protocol
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WGBS was carried out by the Hartwell Center at St. Jude Children’s Research Hospital. Briefly, genomic DNA was extracted using the QIAamp DSP DNA Mini Kit (61304; QIAGEN) and then was bisulfite converted using the EZ-96 DNA Methylation-Gold MagPrep Kit (D5042; Zymo Research). Libraries were prepared from converted DNA using the Accel-NGS Methyl-Seq DNA Library Kit (30096; Swift Biosciences). Libraries were analyzed for insert size distribution with a 2100 BioAnalyzer High Sensitivity Kit (Agilent Technologies), 4200 TapeStation D1000 ScreenTape assay, or Caliper LabChip GX DNA High Sensitivity Reagent Kit (PerkinElmer). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (Life Technologies) or low pass sequencing with a MiSeq nano kit (Illumina). Paired-end 150 cycle sequencing was performed on a NovaSeq 6000 (Illumina).
10
RNA-seq Library Preparation Protocol
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RNA sequencing libraries were prepared using the YourSeq Duet (FT + ’-DGE) RNAseq library kit (Amaryllis Nucleics). Input material was RNA pellets (7.5 μl) from cpSG, which were not quantified beforehand due to the low amount of RNA expected to be present. The full-transcript library preparation protocol was used as described by the manufacturer without modification, except that 19 cycles of PCR were used to amplify the libraries instead of the 14 cycles described in the protocol. Index #1 (ATGATTGA) and index #3 (GCTATTCT) were added to control cpSG RNA samples, and index #2 (GACTGCCT) and index #4 (AGCGTTAC) were added to heat-treated cpSG RNA samples. Prior to sequencing, the prepared libraries were quantified with a Qubit fluorometer (Thermo Fisher) and the Qubit dsDNA HS assay kit to ensure that a sufficient quantity of library (>10 μl of 2 ng/μl per sample) was present. Libraries were pooled and sequenced using the MiSeq Nano kit (Illumina), with 300 bp of paired-end reads (2 × 150 bp) generated. Adapter trimmed reads were mapped to the Arabidopsis genome (Araport11; Cheng et al., 2017 (link)) using the RMTA workflow (v2.6.3; Peri et al., 2019 (link)) with HiSat2 and 0 FPKM filter options.
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