Of note, 1 μg total RNA was prepared for cDNA libraries using the protocol provided by Oxford Nanopore Technologies (ONT). In brief, the SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription and following cDNA PCR for 14 circles with LongAmp Tag (NEB). The PCR products were then subjected to FFPE DNA repair and end-repair (NEB) steps and following adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to FLO-MIN109 flow cells and were run on the PromethION platform at Biomarker Technology Company (Beijing, China).
Longamp tag
Manufactured by New England Biolabs
The LongAmp Tag is a thermal-stable DNA polymerase designed for long-range PCR amplification. It is capable of amplifying DNA fragments up to 20 kb in length.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (10 mentions)
Superscript 4 first strand synthesis system, by Thermo Fisher Scientific (5 mentions)
Cdna pcr sequencing kit sqk pcs109, by Oxford Nanopore (3 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Cdna pcr sequencing kit, by Oxford Nanopore (1 mentions)
Promethion flow cell, by Oxford Nanopore Technologies (1 mentions)
Promethion platform, by Oxford Nanopore Technologies (1 mentions)
Rnasimple total rna kit, by Tiangen Biotech (1 mentions)
Ampure xp beads, by New England Biolabs (1 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (1 mentions)
12 protocols using longamp tag
1
PromethION-based Full-length mRNA Sequencing
2
Profiling Gonad and Skin Transcriptomes
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Total RNA from 4 female gonad, 4 male gonad, 4 head skin of female, and 4 head skin of male samples was respectively extracted. Total RNA with RNA integrity (RIN) number above 7.9 were considered for further analysis (Additional file 3 : Table S3). See Table S1 for additional details. One microgram of total RNA was prepared for cDNA libraries using the cDNA-PCR Sequencing Kit (SQK-PCS109) protocol provided by Oxford Nanopore Technologies (ONT). Briefly, template switching activity of reverse transcriptase enriches full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA. After cDNA PCR for 14 cycles with LongAmp Tag (NEB), the PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to FLO-MIN109 flow cells and run on the PromethION platform at Biomarker Technology Company (Beijing, China).
3
Nanopore-based Full-length Transcriptome Profiling
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A total of 1 µg total RNA was used for cDNA library construction according to the Oxford Nanopore Technology (ONT) protocol. Full‐length mRNAs were reverse transcribed using the SuperScript IV First‐Strand Synthesis System (Invitrogen) and amplified for 14 cycles with LongAmp Tag (NEB). The PCR products underwent the formalin‐fixed paraffin‐embedded DNA repair and end‐repair (NEB) steps followed by adaptor ligation using T4 DNA ligase (NEB). DNA purification was performed using Agencourt XP beads according to ONT protocol. The cDNA libraries were run on a PromethlON platform at the Biomarker Technology Company (Beijing, China). Thresholds for raw reads filtration had a minimum average read quality score of 7 and a minimum read length of 500 bp, and ribosomal RNA was discarded. Full‐length, non‐chimeric transcripts were obtained after mapping to the hg38 genome using minimap2 (version 2.18) and removing redundant transcripts with the cDNA Cupcake package. After polishing with pinfish, consensus isoforms were obtained. Differentially expressed genes were obtained with the DESeq R package (1.18.0), and significantly different expression was filtered out by |Fold change| > 2 and p <0.05. The Nanopore long‐reads RNA‐seq data were deposited in the Gene Expression Omnibus under accession code GSE223088.
4
Nanopore-based Transcriptome Sequencing
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The total RNA of the samples was extracted by a Universal Plant Total RNA Extraction Kit (BioTech, Beijing, China). In total, 1 μg of total RNA was prepared for cDNA libraries using a cDNA-PCR Sequencing Kit (SQK-PCS109, Oxford Nanopore Technologies, Oxford, UK) protocol provided by Oxford Nanopore Technologies (ONT). Briefly, the template switching activity of reverse transcriptase was enriched for full-length cDNAs, and we added defined PCR adapters directly to both ends of the first-strand cDNA. In addition, following cDNA PCR for 14 circles with a LongAmp Tag (NEB), the PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to FLO-MIN109 flowcells and run on the PromethION platform at the Biomarker Technology Company (Beijing, China).
5
Transcriptome Analysis of HUMSC-sEVs Treatment on HCECs
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HCECs (2.5 × 106 cells) were treated with 40 μg/ml HUMSC-sEVs for 48 hours, and the same volume of PBS was added as control with three biological replicates. Total RNA was isolated using the TRIzol reagent according to the manufacturer's instructions. 1 μg total RNA was prepared for cDNA libraries using cDNA-PCR Sequencing Kit (SQK-PCS109) protocol provided by Oxford Nanopore Technologies (ONT). Briefly, the template switching activity of reverse transcriptase enriches for full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA. And it follows cDNA PCR for 14 circles with LongAmp Tag (NEB). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads was used for DNA purification according to ONT protocol. The final cDNA libraries were added to FLO-MIN109 flowcells and run on PromethION platform at Biomarker Technology Company (Beijing, China). The KOBAS software was used to test the statistical enrichment of differential expression transcripts in KEGG pathways.
6
Long-read RNA-seq from FFPE Heart
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Total RNA was extracted from 9 heart samples and prepared for cDNA libraries using the protocol provided by Oxford Nanopore Technologies (ONT). Briefly, SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription and following cDNA PCR for 14 circles with LongAmp Tag (NEB). The PCR products were then subjected to FFPE DNA repair and end-repair (NEB) steps and following adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to ONT protocol. The final cDNA libraries were added to FLO-MIN109 flowcells and were run on the PromethION platform at Biomarker Technology Company (Beijing, China).
7
Transcriptome and Genome Sequencing Protocols
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For transcriptome sequencing, 1 μg of total RNA was used for preparing libraries with cDNA-PCR Sequencing Kit (SQK-PCS109, Oxford Nanopore Technologies). In brief, the full-length cDNAs were enriched using template switching activity of reverse transcriptase. The PCR adapters were directly added to both ends of the first-strand cDNAs. PCR amplification was performed with 14 circles using LongAmp Tag (NEB), and then PCR products were subjected to ONT adaptor ligation using T4 DNA ligase (NEB). The Agencourt XP beads was used for DNA purification according to official protocol. The final cDNA libraries were added to FLO-MIN109 flowcells and run on PromethION platform at Biomarker Technology Company (Beijing, China).
For genome sequencing, the paired-end libraries with 350 bp of insert sizes were constructed according to Illumina's protocol (Illumina, San Diego, CA, USA). In brief, 0.5 μg of genomic DNA was fragmented, end-paired and ligated to adaptors, respectively. After the P2 adapter was added, DNA fragments were fractionated and purified by PCR amplification. Finally, the sequencing libraries were sequenced using Illumina HiSeq2000 at Biomarker Technology Company (Beijing, China).
For genome sequencing, the paired-end libraries with 350 bp of insert sizes were constructed according to Illumina's protocol (Illumina, San Diego, CA, USA). In brief, 0.5 μg of genomic DNA was fragmented, end-paired and ligated to adaptors, respectively. After the P2 adapter was added, DNA fragments were fractionated and purified by PCR amplification. Finally, the sequencing libraries were sequenced using Illumina HiSeq2000 at Biomarker Technology Company (Beijing, China).
8
Peripheral Blood RNA Analysis for Genetic Disorders
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One microgram of total RNA from peripheral blood samples of two affected individuals (F1_II:3 and F1_III:1) and one healthy individual (F1_II:5) was prepared for cDNA libraries using protocol provided by ONT. In brief, SuperScript IV First-Strand Synthesis System (Invitrogen) was used for full-length mRNA reverse transcription and following cDNA PCR for 14 circles with LongAmp Tag (NEB). Agencourt XP beads were used for DNA purification according to ONT protocol. The final cDNA libraries were added to FLO-MIN109 flow cells and were run on PromethION platform at Biomarker Technology Company (Beijing, China).
9
Transcriptomic response to frost stress
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Selected seedlings were exposed to a simulated frost treatment (−2°C) for 0 h, 2 h, and 9 h. Three samples were performed for each cultivar, and each sample contained 12 plants. Leaves at the third and fourth nodes from the tip were collected and stored at −80°C for further analysis.
RNA samples were prepared using an RNA simple Total RNA Kit (DP411, TIANGEN), RNA integrality was tested by agarose gel (LabChip GX, Agient2100), and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific).
Library preparation was performed according to the standard protocol provided by ONT. A mass of 1 μg total RNA was prepared for cDNA libraries using the cDNA-PCR Sequencing Kit (SQK-LSK110+EXP-PCB096) protocol provided by ONT. The template-switching activity of reverse transcriptases enriches full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA, followed by cDNA PCR for 14 cycles with LongAmp Tag (NEB). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to the PromethION Flow Cell (R9 Version, FLO-PRO002, Nanopore) and run on the PromethION platform at Biomarker Technology Company (Beijing, China).
RNA samples were prepared using an RNA simple Total RNA Kit (DP411, TIANGEN), RNA integrality was tested by agarose gel (LabChip GX, Agient2100), and the RNA concentration was detected using a NanoDrop 2000 (Thermo Fisher Scientific).
Library preparation was performed according to the standard protocol provided by ONT. A mass of 1 μg total RNA was prepared for cDNA libraries using the cDNA-PCR Sequencing Kit (SQK-LSK110+EXP-PCB096) protocol provided by ONT. The template-switching activity of reverse transcriptases enriches full-length cDNAs and adds defined PCR adapters directly to both ends of the first-strand cDNA, followed by cDNA PCR for 14 cycles with LongAmp Tag (NEB). The PCR products were then subjected to ONT adaptor ligation using T4 DNA ligase (NEB). Agencourt XP beads were used for DNA purification according to the ONT protocol. The final cDNA libraries were added to the PromethION Flow Cell (R9 Version, FLO-PRO002, Nanopore) and run on the PromethION platform at Biomarker Technology Company (Beijing, China).
10
Arabidopsis Seedling RNA Extraction and Sequencing
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The RNA of Arabidopsis seedlings was extracted by RNAprep Pure Plant Kit (DP441, Tiangen). The cDNA-PCR Barcoding Kit (SQK-PCS109 with SQK-PBK004, Oxford Nanopore Technologies) was used for quality control of total RNA and construction of complementary DNA (cDNA) library. Briefly, the RNA with polyA was reverse transcribed. The cDNA product was amplified for 14 cycles with LongAmp Tag (NEB). Then, adapter addition of cDNA samples was catalyzed by T4 DNA ligase (NEB) and the final cDNA library was purified with Agencourt AMPure XP beads. After the addition of cDNA library to FLO-MIN109 flow cell, sequencing was proceeded by the PromethION platform at Biomarker Technology Company (Beijing, China).
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