The SMARTer Ultra Low Input RNA Kit for Sequencing v4 (Clontech Laboratories, Inc., Mountain View, CA, USA) was used to generate first-strand cDNA from approximately 1 ng of total RNA. Double-stranded cDNA was amplified by long-distance PCR (11 cycles) and purified via magnetic bead clean-up. Library preparation was carried out as described in the Illumina Nextera XT Sample Preparation Guide (Illumina, Inc., San Diego, CA, USA). Input cDNA 150 pg was tagmented (tagged and fragmented) by the Nextera XT transposome. The products were purified and amplified via a limited-cycle PCR program to generate multiplexed sequencing libraries. For the PCR step, 1:5 dilution of the unique dual indexing (i7 and I5) adapters was used. Libraries were quantified using the KAPA Library Quantification Kit–Illumina/ABI Prism User Guide (Roche Sequencing Solutions, Inc., Pleasanton, CA, USA). Equimolar amounts of each library were sequenced on an Illumina NextSeq 2000 instrument controlled by the NextSeq 2000 Control Software (NCS) v1.2.0.36376 using one 50 cycles P3 Flow Cell with the dual index, single-read run parameters. Image analysis and base calling were performed using the Real Time Analysis Software v3.7.17. The resulting .cbcl files were converted into .fastq files with the bcl2fastq v2.20 software.
Nextseq 2000 instrument
Manufactured by Illumina
Sourced in United States
The NextSeq 2000 instrument is a high-throughput sequencing platform designed for a wide range of applications. It utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data. The instrument is capable of sequencing DNA and RNA samples, supporting various library preparation methods and experimental designs.
Automatically generated - may contain errors
Lab products found in correlation
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Smarter ultra low input rna kit for sequencing v4, by Takara Bio (1 mentions)
Nextera xt sample preparation guide, by Illumina (1 mentions)
Qubit 4 fluorometer, by Thermo Fisher Scientific (1 mentions)
Qiaseq stranded mrna select kit, by Qiagen (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Chromium single cell platform, by 10x Genomics (1 mentions)
Luna fl automated fluorescence cell counter, by Logos Biosystems (1 mentions)
Versene, by Thermo Fisher Scientific (1 mentions)
Accutase, by Merck Group (1 mentions)
Qubit 2, by Thermo Fisher Scientific (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Extracol reagent, by EURx (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Respiratory virus oligo panel v2, by Illumina (1 mentions)
Maxwell 16 lev simplyrna purification kit, by Promega (1 mentions)
Cfx384, by Bio-Rad (1 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer rna 6000 pico assay, by Agilent Technologies (1 mentions)
P3 flow cell, by Illumina (1 mentions)
11 protocols using nextseq 2000 instrument
1
SMARTer Ultra Low Input RNA Sequencing
2
Flax Seed Transcriptome Sequencing: cDNA Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
cDNA libraries for sequencing of flax seed transcriptomes were prepared using the QIAseq Stranded mRNA Select Kit (Qiagen) according to the manufacturer’s protocol. RNA from seeds of 10 flax varieties (AGT 422, AGT 427, AGT 981, AGT 1535, Atalante, Entre-Rios, Lola, Norlin, Pechersky kryazh, and Raciol) collected at 3, 7, 14, 21, and 28 DAF from plants grown under three different temperature/watering conditions were used. For 3, 7, 14, and 21 DAF, cDNA libraries were prepared in 2 replicates, and for 28 DAF, cDNA libraries were prepared in 1 replicate (because of the difficulty of obtaining RNA of high quality and sufficient quantity for seeds of late developmental stages). The quality of the obtained cDNA libraries (agreement of the length of the obtained libraries with the expected ones and the absence of adapter dimers) was evaluated on a 2100 Bioanalyzer (Agilent Technologies), and the concentration was evaluated on a Qubit 4 fluorometer (Thermo Fisher Scientific).
The resulting cDNA libraries (266 libraries in total, as the libraries for the variety AGT 422 at 21 and 28 DAF at 24 °C and the variety Lola at 28 DAF at 24 °C were excluded from the analysis because of their low quality) were mixed equimolarly and sequenced on a NextSeq 2000 instrument (Illumina, San Diego, CA, USA) using the NextSeq 2000 P3 Reagents (100 Cycles) kit (Illumina) in 51 + 51 nucleotide format.
The resulting cDNA libraries (266 libraries in total, as the libraries for the variety AGT 422 at 21 and 28 DAF at 24 °C and the variety Lola at 28 DAF at 24 °C were excluded from the analysis because of their low quality) were mixed equimolarly and sequenced on a NextSeq 2000 instrument (Illumina, San Diego, CA, USA) using the NextSeq 2000 P3 Reagents (100 Cycles) kit (Illumina) in 51 + 51 nucleotide format.
3
Dissociation and Single-Cell RNA-seq of hiPSC-derived Islets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hiPSC islets were washed with Versene (Thermo Fisher Scientific, 15040066) and incubated with Accutase (Sigma-Aldrich, A6964-100ML) for 10 min at 37°C. Dissociation was stopped by the addition of PBS supplemented with 1% bovine serum albumin (BSA). Dissociated cells were washed twice in PBS (1%BSA) buffer, centrifuged at 300 rcf for 5 min, filtered using Flowmi 40 μm tip strainer (Bel-Art, H13680-0040), counted and adjusted to 1 × 106 cells ml–1 cells in PBS (1%BSA) for encapsulation. Cell count and viability were measured using the Luna-Fl automated Fluorescence Cell Counter (Logos Biosystems).
For each siRNA treatment, 6000 cells were loaded onto the 10× Chromium Single Cell Platform (10X Genomics) using the Next GEM Single Cell 3′ library and Gel Bead Kit (v3.1 chemistry) according to manufacturer's instructions (10× User Guide CG000204, Revision D). RNA quality was assessed using Tapestation (Agilent). Libraries were quantified using Tapestation (Agilent), the Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) before pooling. Libraries were pooled in equimolar amounts for paired-end sequencing on an Illumina NextSeq 2000 instrument to yield ∼168 million (range 147–195 million) 100-bp-long reads on average per sample.
For each siRNA treatment, 6000 cells were loaded onto the 10× Chromium Single Cell Platform (10X Genomics) using the Next GEM Single Cell 3′ library and Gel Bead Kit (v3.1 chemistry) according to manufacturer's instructions (10× User Guide CG000204, Revision D). RNA quality was assessed using Tapestation (Agilent). Libraries were quantified using Tapestation (Agilent), the Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) before pooling. Libraries were pooled in equimolar amounts for paired-end sequencing on an Illumina NextSeq 2000 instrument to yield ∼168 million (range 147–195 million) 100-bp-long reads on average per sample.
4
CAGE-Seq Transcriptome Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated using an Extracol reagent per the manufacturer’s protocol (EURX, Gdansk, Poland). The quality of total RNA was assessed by Agilent 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States) to confirm that RNA integrity number is over 8.0. Reverse transcription of RNA was performed using random primers (CAGE library preparation Kit; K.K. DNAFORM, Yokohama, Japan). The selection of RNA/cDNA hybrids was enabled by cap-trapping on streptavidin beads. Following the digestion of RNA using RNaseI/H, the linker ligation to 5′ and 3’ cDNA ends enabled the construction of double-stranded cDNA libraries. Sequencing of libraries using the Cap analysis gene expression (CAGE) method was performed using 75 nt single-end reads on a NextSeq 2000 instrument (Illumina, San Diego, CA, United States). Data from this experiment are deposited in Gene expression omnibus (GEO) database (accession number GSE229210). The quality of the obtained data was evaluated via the FastQC tool (v0.11.9). Reads alignment and mapping to a human reference genome (hg38) were performed using the STAR method (Dobin et al., 2013 (link)). Counting tags at each side was performed using SAMtools in R (Rsamtools v2.2.3). In each cell line, CAGE-Seq was performed in biological triplicate for the “WWOX” and “Vec” cellular variants.
5
Respiratory Virus Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples negative for SARS-CoV-2 but collected at timepoints with any reported symptoms of respiratory infection of which we have highlighted: fever, cough, and/or shortness of breath were enriched for respiratory virus nucleic acids using the Illumina Respiratory Virus Oligo Panel v2 (San Diego, California). Libraries were sequenced on an Illumina NextSeq2000 instrument using 2 × 109 paired end reads. Sequences were trimmed of adapter sequences and host reads were removed using bbtools (48 ). Reads were aligned to panel genomes using bowtie2 version 2.2.5 (49 (link)). Consensus sequences were generated using samtools (45 (link)) and iVAR (44 (link)) software packages. Samples with over 1,000 counts and genome-wide coverage were considered positive for infection.
6
Quantitative RT-PCR and RNA-seq Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were reverse transfected, and total RNA was isolated and cDNA synthesized as previously described (13 (link)–15 (link)). Quantitative Real-Time PCR (qRT-PCR) was performed using PowerUp SYBR Green Master Mix (A25778; Thermo Fisher Scientific) on an ABI StepOne Plus Real-Time PCR system (Applied Biosystems, Foster City, CA) or a CFX384 (Bio-Rad, Hercules, CA). Fold-change gene expression was calculated by the ΔΔCT method and normalized to NACA mRNA levels. Samples were generated from three independent experiments unless stated otherwise. For paired-end RNA sequencing (RNA-seq), RNA was extracted (Maxwell 16 LEV simplyRNA purification kit, Promega, Madison, WI), and libraries were prepared and sequenced using standard protocols and a Nextseq 2000 instrument (Illumina, San Diego, CA). The CCR Collaborative Bioinformatics Resource (CCBR) RNA-seq pipeline was used for data analysis https://bioinformatics.ccr.cancer.gov/ccbr/pipelines-software/ccbr-pipeliner/ (see Supplemental Materials and Methods for additional details).
7
RNA-Seq Analysis of HBMEC Cultures
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RNA-Seq analysis, three independent replicates were prepared for each treatment group, Mock, MOI 0.01-, and MOI 0.1-exposed HBMEC cultures, after 6 and 24 h. Total RNA was isolated via the miRNeasy micro kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The RNA was quantified by O.D. measurement before being assessed for quality by chip-based capillary electrophoresis using Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies, St Clara, CA, USA; Part # 5067-1513).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc., San Francisco, CA, USA; Part # 0508-96). The quality and size distribution of the amplified libraries was determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626). Libraries were quantified using the Takara Library Quantification Kit (Shiga, Japan; Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow cell (Illumina, San Diego, CA, USA; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina, San Diego, CA; Part # 20038732) on the NextSeq2000 instrument (Illumina, San Diego, CA, USA).
Libraries were prepared from 150 nanograms (ng) of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc., San Francisco, CA, USA; Part # 0508-96). The quality and size distribution of the amplified libraries was determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies; Part # 5067-4626). Libraries were quantified using the Takara Library Quantification Kit (Shiga, Japan; Part # 638324). The libraries were pooled at equimolar concentrations and diluted prior to loading onto a P3 flow cell (Illumina, San Diego, CA, USA; Part # 20027800) with the P3 300 Cycle reagent kit (Illumina, San Diego, CA; Part # 20038732) on the NextSeq2000 instrument (Illumina, San Diego, CA, USA).
8
Whole Genome Sequencing of L. monocytogenes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Taking into account the sample type, location, and source, L. monocytogenes isolates from all matrices underwent WGS analysis. In the case of stillborn pools, only two out of the four strains were selected, as all isolates displayed identical antimicrobial phenotypes. The extraction of genomic DNA was carried out using the ISOLATE II Genomic DNA kit (Bio-line, London, UK) and quantified with the Qubit fluorometer (Invitrogen, Waltham, MA, USA) using the dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s guidelines. The NexteraXT library preparation protocol (Illumina, San Diego, CA, USA) was applied, and paired-end sequencing (2 × 150 bp) was performed on the NextSeq 2000 instrument (Illumina) following the manufacturer’s instructions.
9
High-throughput 5' RNA sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HT-5PSeq libraries were prepared as previously performed (Zhang and Pelechano, 2021 (link)). Briefly, 6 μg RNA was used for DNase treatment, then DNA-free total RNA were directly ligated with RNA/RNA oligo containing UMI (RNA rP5_RND oligo). Ligated RNA was reverse transcribed and primed with Illumina PE2 compatible oligos containing random hexamers and oligo-dT. RNA in RNA/DNA hybrid was depleted by sodium hydroxide within 20 min at 65 °C incubation. Ribosomal RNAs were depleted using DSN (Duplex-specific nuclease) with the mixture of ribosomal DNA probes. Samples were amplified by PCR and sequenced in an Illumina NextSeq 2000 instrument using 55 sequencing cycles for read 1 and 55 cycles for read 2.
10
RNA-seq Analysis of CD4+ T Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA from isolated CD4+ T cells was extracted and purified using the PicoPure RNA Isolation Kit (Applied Biosystems). Subsequently, RNA-seq libraries were generated as previously described.102 The whole transcriptome amplification (WTA) and tagmentation-based library preparation were performed using Nextera XT (Illumina), followed by sequencing on a NextSeq 2000 Instrument (Illumina). Sequences from RNA-seq were aligned to the human genome (GRCh38) using STAR103 (link) and quantified using RSEM.94 Raw counts at gene or isoform levels were normalized using External RNA Controls Consortium spiked-in controls through RUV-seq,104 (link) and then used for differential gene expression analysis with DESeq2.90 Transcripts per million (TPM) values were used for downstream analysis. Differentially expressed genes (DEGs) from the RNA-seq data set were analyzed using the IPA (Qiagen)105 (link) and the Database for Annotation, Visualization, and Integrated Discovery (DAVID) v6.8.95 (link)
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!