Samples from LNM− primary tumors (n = 4) and LNM+ primary tumors (n = 5) were cut into small specimens. The total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s procedure. The purity and quantity of total RNA were analyzed using NanoDrop ND 1000 (NanoDrop, Wilmington, DE, USA), and the integrity of the RNA was assessed using Agilent 2100 with RIN number >7.0. Poly(A) RNA was purified from total RNA (5 µg) using poly T oligo attached magnetic beads using two rounds of purification (Invitrogen). The mRNA was then fragmented into small pieces using divalent cations under elevated temperature. Subsequently, the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-sequencing sample preparation kit (Illumina, San Diego, CA, USA). Lastly, we performed the 150-bp paired-end sequencing on an Illumina X Ten (LC Bio, Hangzhou, China) following the recommended protocols.
Mrna sequencing sample preparation kit
Manufactured by Illumina
Sourced in United States
The Illumina mRNA Sequencing Sample Preparation Kit is a laboratory equipment product designed for the preparation of mRNA samples for sequencing. The kit provides the necessary reagents and protocols to extract, purify, and prepare mRNA samples for downstream sequencing analysis. The core function of the product is to enable the efficient and standardized preparation of mRNA samples for further processing and sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100 bioanalyzer, by Agilent Technologies (3 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (2 mentions)
Oligo dt magnetic beads, by Illumina (2 mentions)
Rneasy plant mini kit, by Qiagen (2 mentions)
Genome analyzer iix, by Illumina (2 mentions)
X ten, by Illumina (1 mentions)
Poly t oligo attached magnetic beads, by Thermo Fisher Scientific (1 mentions)
Agilent 2100, by Agilent Technologies (1 mentions)
Dynabeads oligo, by Thermo Fisher Scientific (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Novaseq, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Minelute gel extraction kit, by Qiagen (1 mentions)
Genome analyzer iix sequencing platform, by Illumina (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Dna 1000 kit, by Agilent Technologies (1 mentions)
11 protocols using mrna sequencing sample preparation kit
1
RNA-seq Analysis of Lymph Node Metastasis in Tumor Samples
2
RNA-sequencing Protocol for Differential Gene Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA-seq was conducted as previously described (Wang D. et al., 2019 (link)). Total RNA was isolated using TRIzol reagent according to the manufacturer's protocol. Total RNA with RNA integrity number > 7.0 was processed following evaluation using the RNA 6000 Nano LabChip Kit (Agilent, California, USA). Poly (A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (Salajegheh et al.) 25–61005 (Thermo Fisher, CA, USA) by two rounds of purification and then fragmented under the following conditions: 94°C for 5–7 min. The cleaved RNA fragments were reverse transcribed to create a final cDNA library according to the manufacturer's protocol (mRNA Sequencing Sample Preparation Kit, Illumina). The average insert size for the cDNA library was 300 ± 50 bp. We performed 2 × 150 bp end sequencing (PE150) on an Illumina Novaseq 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) according to the manufacturer's instructions. Differential expression analysis was performed based on adjusted p-values; volcano plots were prepared to depict fold-change differences in gene expression. Gene Ontology (GO) enrichment and Kyoto Encyclopedia for Genes and Genome (KEGG) analyses of the differentially expressed mRNAs were also conducted. RNA-sequencing data were submitted to Gene Expression Omnibus (GEO) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161737 ].
3
RNA Isolation and mRNA-Seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A portion (100 mg) of each sample was ground to powder in liquid nitrogen using a mortar and pestle. Total RNA was isolated using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). The quantity and quality were determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The poly (A) mRNA was isolated from total RNA samples with Magnetic Oligo (dT) Beads (Illumina, San Diego, CA, USA) and used for mRNA-Sequencing library construction. cDNA was synthesized using the fragmented mRNA and end-repaired followed by a single ‘A' base addition. mRNA-Sequencing Sample Preparation Kit (Illumina) was used to prepare the DNA fragments for ligation to the adapters. After purification, the cDNA fragments (200 ± 25 bp) were excised and retrieved. PCR was performed to selectively enrich and amplify the cDNA fragments. Libraries were prepared from a 150–200 bp size-selected fraction following adapter ligation and agarose gel separation.
4
Liver RNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total liver RNA was extracted by TRIzol reagent following the manufacturer’s instructions (Invitrogen, Carlsbad, CA). The quantity and purity of RNA were analyzed using Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit (Agilent, Folsom, CA). After purification, the poly (A) - or poly (A) + RNA fractions was fragmented into small pieces by using divalent cations under increased temperature. Then, the cleaved RNA fragments were reverse-transcribed to create the final complementary DNA library in accordance with the protocol for the mRNA-sequencing sample preparation kit (Illumina, San Diego, CA), the average insert size for the paired-end libraries was 300 bp (±50 bp).34 (link) Then, the paired-end sequencing on an Illumina Novaseq was performed at the Berry Genomics Corporation (Beijing, China) following the vendor's recommended protocol.
5
RNA Isolation and mRNA-Seq Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation and library construction were performed according to the method described by Yang et al. (2017) [27 (link)]. In brief, a portion (100 mg) of each sample was ground to powder in liquid nitrogen. Total RNA was isolated using RNeasy Plant Mini kit (Qiagen, Hilden, Germany). The quantity and quality of RNA were determined using Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). The poly (A) mRNA was isolated from total RNA samples with Magnetic Oligo (dT) Beads (Illumina, San Diego, CA, USA) and used for mRNA-sequencing library construction. cDNA was synthesized using the fragmented mRNA with the incorporation of reverse transcriptase for end-repair, followed by a single ‘A’ base addition. mRNA-Sequencing Sample Preparation Kit (Illumina) was used to prepare the DNA fragments for ligation to the adapters. After purification, the cDNA fragments (200 ± 25 bp) were excised and retrieved. Polymerase chain reaction (PCR) was performed to selectively enrich and amplify the cDNA fragments. Libraries were prepared from a 150–200 bp size-selected fraction following adapter ligation and agarose gel separation.
6
RNA-seq Analysis of Sorted Lin⁻c-kit⁺ HSPCs
7
Deep Sequencing of Mouse Transcriptome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolation of mRNA, cDNA synthesis, and preparation of libraries for deep sequencing were performed with mRNA Sequencing Sample Preparation Kit (Illumina), NEBNext mRNA Library Preparation Reagent Set for Illumina (New England Biolabs), QIAquick PCR Purification Kit (Qiagen) and MinElute Gel Extraction Kit (Qiagen) according to the standard protocols. The sequencing procedure (single-end 72 nucleotide reads with Sequencing Control Software (SCS) v. 2.10 and Real Time Analysis (RTA) software v. 1.8) was carried out in a Genome Analyzer IIx (Illumina) as recommended by the manufacturer. The FASTQ Illumina reads have been filtered with fastq_quality_filter to select the reads with a quality of 25 or higher for each letter. These reads were aligned to the whole mouse transcriptome sequences (ftp://ftp.ncbi.nih.gov/genomes/M_musculus/RNA/ ) with the BWA program using parameters “bwa index -a is” and “bwa aln -N -t 8”. The results were used to calculate the transcripts reads coverage statistics for each RNA sample. The relative abundance of each mRNA was calculated in Microsoft Excel as the number of reads aligned to this mRNA divided by the total number of reads aligned to the mouse transcriptome.
8
Transcriptome Analysis of Plant Development
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from frozen mother scales and bulblets separately according to Li et al. [47 ]. The purity and concentration of total RNA were determined using an Infinite® 200 PRO (Tecan, Männedorf, Switzerland) as well as RNA gel electrophoresis (formaldehyde buffer system). The cDNA libraries of 0 d, 15 d and 35 d were constructed using the mRNA Sequencing Sample Preparation Kit following the manufacturer’s (Illumina, CA, USA) instructions. cDNA fragments 200 ± 25 bp in size were selected for PCR amplification. Finally, sequencing was performed on the Illumina cluster station and Illumina Genome Analyzer IIx sequencing platform. The clean reads generated were then used for all subsequent analyses. Trinity (http://trinityrnaseq.sourceforge.net/ ) was used to assemble the pair-end short reads into contigs.
9
RNA-seq Analysis of Biliary Atresia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pooled RNA libraries were prepared using Illumina’s mRNA Sequencing Sample Preparation kit (#RS-930-1001) from the explanted liver tissue samples of 6 BA patients and 6 normal pre-implant liver allografts. The pooled RNA libraries for the BA and the normal groups were sequenced on Genome Analyzer II with 36 bp paired-end at the Wistar Institute, Philadelphia. CASAVA software was used to generate a table of exon, gene, and splice counts in both BA and normal groups. DEseq, R package for RNAseq data, was used for differential analysis (Anders and Huber, 2010 (link)). The genes among the 40% lower quantile of the total read counts were removed to maximize the statistical power while maintaining the majority of the differentially regulated genes when the statistical analysis was performed without the removal. For variance estimation, the DEseq options of “blind” and “fit-only” were used. The resulting dispersion plot was checked for quality. The enrichment analysis of differentially regulated genes under the adjusted p-value cutoff of 0.1 using Benjamini-Hochberg method was performed using DAVID (Huang et al., 2009 (link)). EASE score, a modified Fisher exact p-value for DAVID enrichment, of 0.05 was used for the statistical cutoff.
10
Paired-End RNA-Seq of PC-3 and Ep156T Xenografts
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Paired-end high-throughput RNA sequencing of PC-3 xenografts, Ep156T xenografts and intact samples was performed on the Illumina/Solexa GAII platform (Illumina Inc, San Diego, CA, USA) by the ServiceXS Sequencing Facility (Leiden, Netherlands).
The Illumina mRNA sequencing sample preparation kit was used to process all RNA samples. In brief, after isolation of mRNA from total RNA using poly-T-oligo-attached magnetic beads, mRNA was fragmented (using divalent cations), which yielded an average size of 200 nucleotides (fragment library). Following cDNA synthesis, cDNA was ligated with adapters and amplified by PCR. The quality and yield was evaluated using a DNA 1000 kit (Agilent Technologies). cDNA fragments were hybridized onto an Illumina glass flow cell, fragments were amplified into a cluster using bridge amplification. 8pmol of DNA per sample was sequenced. Two sequencing reads of 75 cycles each using the Read 1 sequencing and Read 2 sequencing primers were performed using a glass flow cell. Sequencing files were generated in FastQ format.
The Illumina mRNA sequencing sample preparation kit was used to process all RNA samples. In brief, after isolation of mRNA from total RNA using poly-T-oligo-attached magnetic beads, mRNA was fragmented (using divalent cations), which yielded an average size of 200 nucleotides (fragment library). Following cDNA synthesis, cDNA was ligated with adapters and amplified by PCR. The quality and yield was evaluated using a DNA 1000 kit (Agilent Technologies). cDNA fragments were hybridized onto an Illumina glass flow cell, fragments were amplified into a cluster using bridge amplification. 8pmol of DNA per sample was sequenced. Two sequencing reads of 75 cycles each using the Read 1 sequencing and Read 2 sequencing primers were performed using a glass flow cell. Sequencing files were generated in FastQ format.
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!