Three biological replicates of 9464D and M1 cells were collected, and total RNA isolation was done using a TRIzol reagent (Invitrogen, Carlsbad, CA, USA), as per the manufacturer’s protocol, before being subjected to RNA-seq. RNA-seq libraries were prepared in the Penn State College of Medicine Genome Sciences core (RRID: SCR_021123) using the Illumina Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA), as per the manufacturer’s instructions. Briefly, polyA RNA was purified from 200 ng of total RNA using oligo (dT) beads. The extracted mRNA fraction was subjected to fragmentation, reverse transcription, end repair, 3′– end adenylation, and adaptor ligation, followed by PCR amplification and magnetic bead purification (Omega Bio-Tek, Norcross, GA, USA). The unique dual index sequences (IDT® for Illumina RNA UD Indexes Set C, Ligation, Illumina) were incorporated into the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using a BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA). The libraries were pooled and sequenced via Illumina NovaSeq 6000 (Illumina) to get, on average, 25 million paired-end 50 bp reads, according to the manufacturer’s instructions.
Stranded mrna prep ligation kit
Manufactured by Illumina
Sourced in United States
The Stranded mRNA Prep Ligation kit is a laboratory equipment product designed for the preparation of stranded mRNA samples. It facilitates the ligation of sequencing adapters to mRNA molecules, a crucial step in the mRNA sequencing workflow. The kit provides the necessary reagents and protocols to enable the generation of sequencing-ready mRNA libraries.
Automatically generated - may contain errors
Lab products found in correlation
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Novaseq 6000, by Illumina (3 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (3 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 sequencing device, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Ercc spike in, by Thermo Fisher Scientific (1 mentions)
Direct zol rna microprep kit, by Zymo Research (1 mentions)
Nextseq 2000 device, by Illumina (1 mentions)
Oligo dt beads, by Illumina (1 mentions)
Phaselock tubes, by Quantabio (1 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Qiazol lysis reagent, by Qiagen (1 mentions)
Rneasy lipid tissue mini kit, by Qiagen (1 mentions)
Nanodrop 1000, by Agilent Technologies (1 mentions)
Rna 6000 nano, by Agilent Technologies (1 mentions)
2200 tapestation system, by Agilent Technologies (1 mentions)
13 protocols using stranded mrna prep ligation kit
1
RNA-seq analysis of 9464D and M1 cells
2
Illumina Stranded mRNA Sequencing
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RNA-seq library preparation was performed with Illumina’s Stranded mRNA Prep Ligation Kit following the Stranded mRNA Prep Ligation Reference Guide (June 2020) (document no. 1000000124518 v00). Libraries were profiled on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851) in a Qubit 2.0 Fluorometer (Life Technologies), following the manufacturer’s recommended protocols. Samples were pooled in equimolar ratios and sequenced on an Illumina NextSeq 500 sequencing device with one or two dark cycles upfront as 79-, 80- or 155-nt single-end reads.
3
RNA Extraction and NGS Library Preparation
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RNA was extracted using TRIzol (Fisher Scientific, 15-596-026) and a Direct-zol RNA MicroPrep Kit (Zymo Research, R2062). For pupa samples, only the aqueous phase formed after phenol–chloroform separation was loaded on the column after mixing with 100% ethanol. NGS library preparation was performed using an Illumina Stranded mRNA Prep Ligation kit according to the Stranded mRNA Prep Ligation Reference Guide (June 2020; document no. 1000000124518 v00). For the Ag55 cell culture RNA-seq, libraries were prepared with a starting amount of 100 ng and 2 μl of ERCC spike-ins (Ambion, 4456740) in a 1:1,000 dilution and amplified in 12 PCR cycles. For the pupa RNA-seq, libraries were prepared with a starting amount of 1,000 ng and 2 μl of ERCC spike-ins (Ambion, 4456740) in a 1:100 dilution and amplified in 10 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies), and quantified using a Qubit dsDNA HS Assay kit in a Qubit 2.0 Fluorometer (Life Technologies). Pooled samples were sequenced on a NextSeq 500 High Output, PE for 2× 73 cycles plus 2× 10 cycles for the dual index read.
4
High-throughput RNA-Seq library preparation
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Library preparation and Illumina sequencing were performed at the Ecole Normale Supérieure Genomique, ENS core facility (Paris, France). Messenger (polyA+) RNAs were purified from 200 ng of total RNA using oligo(dT). Libraries were performed using the strand-specific RNA-Seq library preparation Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA) and were multiplexed by 26 to 28 on 5 P3 flowcells and on additional a P2 flowcell with 6 samples (Illumina). A 68 bp single-end read sequencing was performed on a NextSeq 2000 device (Illumina). A mean of 41 ± 12 million passing Illumina quality filter reads was obtained for each of the 12 samples.
5
Conjunctival Transcriptome RNA-Seq Protocol
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Conjunctival tissue samples were collected into tubes containing RNAlater and were later homogenized using QIAzol lysis reagent (Qiagen) and ceramic beads (Precellys). Phase-lock tubes (Quantabio) were used to assist separation following chloroform-based phase separation of RNA before total RNA was extracted with the RNeasy Lipid Tissue Mini Kit (Qiagen). RNA quantification and quality assessment were performed with Nanodrop 1,000 and RNA 6,000 Nano (Agilent Bioanalyzer). Total RNA was diluted to 1 μg in 25 μL of RNase-free water prior to a polyA selection using Oligo(dT) beads from Illumina’s Stranded mRNA Prep Ligation kit. Enriched mRNA species were converted to full length complementary deoxyribonucleic acid, dual indexed, and then amplified using 10 PCR cycles. The final libraries were assessed using the D1000 kit on the TapeStation (Agilent) for quality as well as the High Sensitivity DNA Qubit for quantification. To prepare for sequencing, libraries were pooled together at equal molar before denaturing with sodium hydroxide and diluted to 1.5 pM in accordance with Illumina’s denaturation protocol for the NovaSeq6000. Sequence runs were performed on a NovaSeq6000 V1.5 SP flow cell and a 2′ 90 bp paired-end run.
6
CD3+ Cell Transcriptome Profiling
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Total RNA was isolated from bead selected CD3+ cells from patient PBMC using Qiagen’s AllPrep DNA/RNA Mini kit per manufacturer’s instructions. RNA quantity and quality were assessed with the Qubit 4 Fluorometer (ThermoFisher) and the 2200 TapeStation system (Agilent), respectively. RNAseq libraries were prepared with Illumina’s Stranded mRNA Prep Ligation kit, starting with 100 ng of total RNA. This method uses oligo(dT) magnetic beads to capture mRNAs with polyA tails. Paired-end sequencing was performed using the NovaSeq 6000 version 1.0 (2 × 100).
7
RNA-seq Analysis of Hippocampal Tissue
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RNA from snap-frozen injected whole hippocampus tissue was quality controlled using Agilent 2100 Bioanalyzer and RNA quantity was detected by Qubit fluorometric quantitation. Samples were sequenced at a minimum RIN value of 8.0. Libraries were performed using the Illumina Stranded mRNA Prep, Ligation kit. High-throughput sequencing was performed using an NS500HO flowcell (Illumina) for an average read of ∼16×106 reads. Fastq files were assessed using bcl2fastq/QC report. Sequencing was performed at the Genomics Core Facility at NTNU.
8
Isolation and Sequencing of Peripheral Blood Leukocytes
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The regular examination included the complete blood count test, obtaining 5 mL of EDTA-treated blood via venepuncture, and other tests at the Central Laboratory. An aliquot of 1.5 mL was saved and prepared for leukocyte isolation. A whole blood specimen was used to isolate peripheral white blood cells using a QIAMP® RNA Blood kit. The RNA quantity was determined using a Qubit® 4 Fluorometer. The isolated RNA was kept at −80 °C. The Illumina® Stranded mRNA Prep Ligation Kit was used to prepare all samples for library preparation. Following preparation, the samples were maintained at −20 °C. Nextseq 550 Illumina® was used for RNA-seq.
9
RNA Extraction and RNA-seq from Brain Regions
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RNA was extracted from two brain regions (ACC and HP) using the PureLink® RNA Mini Kit (Ambion #12183018A) according to the manufacturer’s instructions. The quantity and the integrity of isolated RNA samples were evaluated using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). RNA-seq libraries were prepared with an Illumina® Stranded mRNA Prep, Ligation Kit (Illumina #20040532) and IDT® for Illumina® RNA UD Indexes Set A, Ligation (Illumina #20040553). The RNA library concentration was measured using a Qubit® 2.0 fluorometer and successfully sequenced on an Illumina NovaSeq6000 (Illumina Inc., San Diego, CA, USA).
10
High-Quality RNA Sequencing Pipeline
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RNA extraction was performed as per RNA/DNA Purification Kit instructions (Norgen, Thorold, Canada). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent, Santa Clara, California, USA) and the kit RNA 6000 Pico Kit Quick Start (Agilent, California, USA). For each condition, the four replicates with the best RNA quality were used to create 12 libraries with the Stranded mRNA Prep Ligation kit (Illumina, San Diego, California, USA), and a pre-run on a MiniSeq (mid-output flow cell) and a NovaSeq run (S4 flow cell) were performed on the MGX facility (https://www.mgx.cnrs.fr/ ).
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