The primed plate was filled with cells at a concentration of 800–1000 cells/μL. To initiate reverse transcription, the plate was placed on a thermomixer and incubated at 37 °C and 1200 rpm for 20 min. Afterward, Exonuclease I was added to the plate and further incubated on the thermomixer for 30 min. The plate was then transferred to a heat block and incubated at 80 °C for 20 min. The cDNA library was prepared using the BD protocol, following the instructions provided by Vallejo et al. Finally, the quality control and quantification assessments of the cDNA library were conducted using the TapeStation, Qubit kits, and associated reagents (Thermo Fisher, Waltham, MA, USA).
Qubit kits
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Qubit kits are a series of fluorometric assays designed to accurately quantify DNA, RNA, and protein samples. The kits utilize fluorescent dyes that bind specifically to the target molecules, allowing for precise measurements of the sample concentrations.
Automatically generated - may contain errors
Lab products found in correlation
Miseq machine, by Illumina (2 mentions)
Bioanalyzer dna1000 chips, by Agilent Technologies (2 mentions)
16s metagenomic sequencing library preparation, by Illumina (2 mentions)
Tapestation, by Thermo Fisher Scientific (1 mentions)
Glycogen, by Roche (1 mentions)
Tlrl eblps, by InvivoGen (1 mentions)
Tlrl pma, by InvivoGen (1 mentions)
Tlrl pic, by InvivoGen (1 mentions)
Ionomycin inh ion, by InvivoGen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
4 protocols using qubit kits
1
Single-cell cDNA Library Preparation
2
16S rRNA Amplicon Sequencing of Microbial Genomes
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Amplicon PCR was performed with microbial genomic DNA using a concentration of 5 ng/µl in 10 mM Tris pH 8.5. PCR amplification of the V3/V4 regions of bacterial 16S rRNA encoding gene was carried out using the primers Pro341-XT (TCG-TCG-GCA-GCG-TCA-GAT-GTG-TAT-AAG-AGA-CAG-CCT-ACG-GGN-BGC-ASC-AG) and Pro805-XT (GTC-TCG-TGG-GCT-CGG-AGA-TGT-GTA-TAA-GAG-ACA-GGA-CTA-CNV-GGG-TAT-CTA-ATC-C) which resulted in amplicon sizes smaller than 550 bp. The details of library constructions, such as index PCR, PCR clean-up 2, library quantification, normalization, and pooling were performed corresponding to the Illumina “16S Metagenomic Sequencing Library Preparation” protocol.
Bioanalyzer DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and Qubit kits (Thermo Fischer Scientific, Waltham, MA, USA) were applied for quantity and quality controls of each individual sample library and the final library pool; 5 pM of the final library mixture, containing at least 5% PhiX control, was exposed to one individual sequencing run using a 2x250 or 2x300 cycle 3 reagent cartridge on an Illumina MiSeq machine. All raw data fastq files were used for sequence data analyses.
Bioanalyzer DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and Qubit kits (Thermo Fischer Scientific, Waltham, MA, USA) were applied for quantity and quality controls of each individual sample library and the final library pool; 5 pM of the final library mixture, containing at least 5% PhiX control, was exposed to one individual sequencing run using a 2x250 or 2x300 cycle 3 reagent cartridge on an Illumina MiSeq machine. All raw data fastq files were used for sequence data analyses.
3
Amplicon PCR of Microbial 16S rRNA Gene
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Amplicon PCR was performed with microbial genomic DNA using concentrations of 5 ng/μl in 10 mM Tris pH 8.5. PCR amplification of the V3/V4 regions of bacterial 16S rRNA encoding gene was carried out using the primers Pro341-XT (TCG-TCG-GCA-GCG-TCA-GAT-GTG-TAT-AAG-AGA-CAG-CCT-ACG-GGN-BGC-ASC-AG ) and Pro805-XT (GTC-TCG-TGG-GCT-CGG-AGA-TGT-GTA-TAA-GAG-ACA-GGA-CTA-CNV-GGG-TAT-CTA-ATC-C ) which resulted in amplicon sizes smaller than 550bp. The details of library constructions, such as Index PCR, PCR clean-up 2, library quantification, normalization and pooling were performed corresponding to the Illumina “16S Metagenomic Sequencing Library Preparation” protocol.
Bioanalyzer DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and Qubit kits (Thermo Fischer Scientific, Waltham, MA, USA) were applied for quantity and quality controls of each individual sample library and the final library pool. 5 pM of the final library mixture, containing at least five percent PhiX control, were exposed to one individual sequencing run using a 2x250 or 2x300 cycle 3 reagent cartridge on an Illumina MiSeq machine. All raw data fastq files were used for sequence data analyses. The raw sequencing fastq files will be submitted to the BioProject database.
Bioanalyzer DNA 1000 chips (Agilent Technologies, Santa Clara, CA, USA) and Qubit kits (Thermo Fischer Scientific, Waltham, MA, USA) were applied for quantity and quality controls of each individual sample library and the final library pool. 5 pM of the final library mixture, containing at least five percent PhiX control, were exposed to one individual sequencing run using a 2x250 or 2x300 cycle 3 reagent cartridge on an Illumina MiSeq machine. All raw data fastq files were used for sequence data analyses. The raw sequencing fastq files will be submitted to the BioProject database.
4
Stimulation and RNA Extraction from Splenocytes
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Splenocytes were rested in complete IMDM (cIMDM) (Sigma) containing 10% FBS, 10 U/ml Penicillin and 10 μg/ml Streptomycin, 40 μM β-Mercaptoethanol and 10 mM HEPES. Following centrifugation, splenocytes were re-suspended and 8.5–10 × 105 cells were seeded in 96-well flat-bottom plates. Following overnight culture at 37°C, splenocytes were stimulated with an agonist array; either 10 μg/200 μl lipopolysaccharide (LPS) from Escherichia Coli 011:B4 (tlrl-eblps, Invivogen), 10 μg/200 μl Concanavalin A (ConA) (C0412, Sigma), 1 μg/200 μl Phorbol myristaste acetate (PMA) (tlrl-pma, Invivogen) mixed with 10 μg/200 μl Ionomycin (inh-ion, Invivogen) or transfected with 1 μg/200 μl high molecular weight polyinosinic:polycytidylic acid (poly I:C) (tlrl-pic, Invivogen) using 3 μl Lipofectamine 3000 (L3000001, LifeTechnologies). After 6 h, RNA was extracted with use of TRIzol Reagent (10296028, Invitrogen) according to manufacturer's instructions. The RNA pellet was visualized by the addition of 1 μl of glycogen (10901393001, Roche) and re-suspended in 40 μl RNase free water. RNA yields ranged from 20 to 25 ng/μl as measured with high sensitivity Qubit kits (Q32852, ThermoFisher).
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