Total RNA was isolated from proliferating and differentiated CPCs using the RNeasy isolation kit (Qiagen, Valencia, California). Sequencing libraries were prepared according to Illumina RNA Seq library kit instructions with Poly(A) selection (Illumina, San Diego, California). Libraries were sequenced with the Illumina HiSeq2000 (2 × 100 bp).
Poly a selection
Manufactured by Illumina
Poly(A) selection is a laboratory technique used to isolate and enrich messenger RNA (mRNA) from a mixed population of RNA molecules. The core function of this equipment is to selectively capture and purify the polyadenylated (poly(A)) tail present at the 3' end of mRNA, allowing for the separation of mRNA from other non-polyadenylated RNA species such as ribosomal RNA and transfer RNA.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy isolation kit, by Qiagen (2 mentions)
Rna seq library kit, by Illumina (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Sv total rna isolation system, by Promega (1 mentions)
Truseq stranded mrna library preparation kit, by Illumina (1 mentions)
Hiseq system, by Illumina (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
6 protocols using poly a selection
1
RNA Sequencing of Proliferating and Differentiated CPCs
2
Transcriptional Profiling of HA-TgFBXL2 Parasites
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HATgFBXL2 parasites were grown in the presence of ATC for either 24 or 48 h. HATgFBXL2 parasites growing in the absence of ATC were used as control. After ATC treatment, parasites were harvested from infected HFF monolayers by scraping and passage through a 27-gauge needle, centrifuged at 2000 × g for 8 min at room temperature, resuspended in phosphate-buffered saline, counted, pelleted and total RNA was extracted using SV Total RNA Isolation System (Promega; Madison, WI).
Agilent 2100 Bioanalyzer was used to determine the integrity, purity, and concentration of RNA samples. RNA integrity (RIN) score of 6.5 or above was considered acceptable for further analysis. Total RNA was enriched for mRNA using poly-(A)-selection (Illumina; San Diego, CA). NEB stranded RNA library prep kit (NEB) and NEB Ultra II RNA library prep kit (NEB) were used to prepare complementary DNA (cDNA) libraries for all HATgFBXL2 samples, according to manufacturer’s protocol. RNA sequencing was carried out on an Illumina HiSeq2500 (Illumina) with a mid-output 75-cycle paired end with 10–20 million reads per sample at the Genomics and Bioinformatics core facility at the University at Buffalo.
Agilent 2100 Bioanalyzer was used to determine the integrity, purity, and concentration of RNA samples. RNA integrity (RIN) score of 6.5 or above was considered acceptable for further analysis. Total RNA was enriched for mRNA using poly-(A)-selection (Illumina; San Diego, CA). NEB stranded RNA library prep kit (NEB) and NEB Ultra II RNA library prep kit (NEB) were used to prepare complementary DNA (cDNA) libraries for all HATgFBXL2 samples, according to manufacturer’s protocol. RNA sequencing was carried out on an Illumina HiSeq2500 (Illumina) with a mid-output 75-cycle paired end with 10–20 million reads per sample at the Genomics and Bioinformatics core facility at the University at Buffalo.
3
RNA-Seq Analysis of Mouse Samples
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Sequencing library preparation was performed using TruSeq stranded mRNA library preparation kit with poly A selection (Illumina Inc.). Cluster generation and paired-end sequencing was performed 125 cycles in one lane by Illumina HiSeq system, executed by SNP&SEQ Technology Platform at Uppsala Biomedical Centre. Base calls were converted to fastq format. Trimmomatic command was used to remove TruSeq3 adaptors. Quality control was performed using multiQC (see: multiqc_report_afterTrimmingAdaptors.html for QC report). Trimmed reads were mapped to mm10 (mouse) reference genome and quantified using Salmon. Differential expression analysis was performed using DESeq2 (R package). An FDR adjusted threshold of 0.005 was used to discriminate significantly regulated proteins.
Samples were clustered using hierarchical dendrogram and principal component analysis (PCA; Extended DataFig. 1-2 ). Whereas most samples clustered according to genotype, two samples (WT3 and KO5) extremely deviated from other samples and these outliers were excluded.
Samples were clustered using hierarchical dendrogram and principal component analysis (PCA; Extended Data
4
Allele-Specific Expression Analysis of Horse Liver Transcriptome
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Our validation set consisted of short-read RNA-seq data from 66 liver samples, 41 males and 26 females, with no evidence of liver disease. These samples were comprised of 12 breeds including 27 Quarter Horses, 17 Warmbloods, 8 Thoroughbreds, 3 Andalusians, 3 Arabians, 2 Lusitanos, 1 Percheron, 1 Shire, 1 Ponys of the Americas, 1 Friesian, 1 Mustang, and 1 Gypsy Vanner. The average age of this cohort was 3.49 years and ranged from 1 month to 8 years of age. RNA-seq was performed with Illumina polyA-selection with a read length of 2 × 150bp. Adapter trimming, poly-A trimming, N trimming, quality trimming, length filtering, and removal of PCR duplicates were conducted using HTStream, version 1.3.3.26 Reads were aligned to EquCab.3.0 using STAR, version 2.7.10b.27 (link) Expression counts at identified ASE loci in liver were generated using SAMtools mpileup from SAMtools 1.18.24 (link) Loci that were not heterozygous were filtered out. We used the same significance criteria as our original cohort - aeFC ≥ 2 and adjusted p-value ≤ .05
5
RNA Sequencing of Proliferating and Differentiated CPCs
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Total RNA was isolated from proliferating and differentiated CPCs using the RNeasy isolation kit (Qiagen, Valencia, California). Sequencing libraries were prepared according to Illumina RNA Seq library kit instructions with Poly(A) selection (Illumina, San Diego, California). Libraries were sequenced with the Illumina HiSeq2000 (2 × 100 bp).
6
Transcriptome Profiling of Brain Tissue
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Twenty brains were used for each replicate (three biological replicates/condition). Brains were dissected and put into Trizol (Thermo Fischer Scientific). RNA was isolated using a standard Trizol protocol (Goodman et al., 2019 (link)). RNA was purified using RNA Clean & Concentrator Kit R10114 (Zymo Research, Irvine, CA). RNA sequencing by Admera Health BioPharma Services (South Plainfield, NJ), using TruSeq Stranded mRNA Kit with PolyA selection (Illumina; San Diego, CA).
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