The mRNA library preparation followed the SQK-RNA002 kit (Oxford Nanopore)–recommended protocol; the only modification was the input mRNA quantity increased from 500 to 1,000 ng, and all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermo Fisher Scientific, Q32851) with minimum RNA preparations reaching at least 200 ng. For all conditions, sequencing was carried out on FLO-MIN106 flow cells using either a MinION MK1C or MinION sequencer. All datasets were subsequently base called (high-accuracy base calling) with a Guppy version higher than 5.0.1 with a Q score cutoff of >7. Long-read alignment was carried out by Minimap2 as previously described49 (link). Sam files were converted to bam and sorted using Samtools 1.4. Alignments were converted and sorted using Samtools 1.4.1. For the three described samples, Toxoplasma aligned reads range between 600,000 and 800,000. The Nanopore direct RNA-seq dataset is available at the National Center for Biotechnology Information: BioProject number PRJNA921935 .
Sqk rna002 kit
Manufactured by Oxford Nanopore
Sourced in United Kingdom
The SQK-RNA002 kit is a laboratory product manufactured by Oxford Nanopore. It is designed to enable RNA sequencing using Oxford Nanopore's nanopore technology. The kit contains the necessary reagents and consumables for sample preparation and sequencing. The core function of the SQK-RNA002 kit is to facilitate the analysis of RNA molecules through nanopore-based sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Min106d r9 version flow cells, by Oxford Nanopore (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (3 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (2 mentions)
Promethion, by Oxford Nanopore Technologies (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
R9.4.1 flow cell, by Oxford Nanopore (1 mentions)
Escherichia coli poly a polymerase, by New England Biolabs (1 mentions)
Qubit dna hs assay kit, by Thermo Fisher Scientific (1 mentions)
R9.4 flow cell, by Oxford Nanopore (1 mentions)
Agencourt ampure xp magnetic beads, by Beckman Coulter (1 mentions)
Minion mk1c sequencer, by Oxford Nanopore (1 mentions)
Superscript 3 reverse, by Thermo Fisher Scientific (1 mentions)
13 protocols using sqk rna002 kit
1
Nanopore Direct RNA-Seq Protocol
2
Direct RNA Sequencing of Mouse Brain
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RNA was sequenced directly using a Nanopore PromethION instrument. Poly(A) RNA was isolated from mouse brain cortex (WT and Fmr1 KO, both C57BL/6J) using µMACS mRNA Isolation kit (130-075-101). Five hundred nanograms was used for library preparation for direct RNA sequencing with SQK-RNA002 kit (Oxford Nanopore). Sequencing was performed using PromethION with the FLO_PRO002 flow cell (Oxford Nanopore). Base calling of the Fast5 data outputs from PromethION was performed using Guppy (v4.0.11) and then converted into Fastq files. Reads were aligned to the reference mouse transcriptome mm10 (Ensembl) using Minimap2 (v2.17) with the following flags: “-ax map-ont -N 100”, and only primary alignments were kept. Sorting and indexing of reads were performed using Samtools (v1.9). Poly(A) length estimation was performed using Nanopolish (v0.13.2) using the default setting. Transcripts with less than 10 reads per condition were filtered out. t-tests were used on average poly(A) length in each sample to identify statistically significant genotype-dependent poly(A) length changes of at least 20 residues (P < 0.05). Isoform quantification was performed using Salmon (v1.5.2) in alignment-based mode and differential isoform expression was performed using DESEQ2. Nanopore sequencing was performed with biologic triplicates.
3
Direct RNA Sequencing of Sindbis Virus
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Poly(A)+ RNAs were enriched from total RNAs using Dynabeads mRNA Purification Kit (Invitrogen). IVT RNAs were polyadenylated using Escherichia coli poly(A) polymerase (New England Biolabs). A total of 1,000 ng of poly(A)+ RNAs were subjected to DRS library preparation using an SQK-RNA002 kit (Oxford Nanopore Technologies, Oxford, UK). The optional reverse transcription step was included using SuperScript III Reverse Transcriptase (Invitrogen). Sequencing was performed on the MinION platform using R9.4.1 flow cells (Oxford Nanopore Technologies).
Reads were base called using the Guppy workflow (v5.0.16) with the configuration of rna_R9.4.1_70bps_hac.cfg or using the Dorado workflow (v0.3.4) with the configuration of rna002_70bps_hac@v3. The resulting FASTQ files were aligned to the SINV genome (GenBank:NC_001547.1 ) using Minimap2 v2.17 with parameter settings “-ax map-ont” (51 (link)). Mapping results were stored in SAM files, which were subsequently converted into bam files and sorted and indexed using SAMtools v1.17 (52 (link)). Read alignment information was extracted using a custom Python script based on Pysam. Full-length viral reads, defined as those covering ≥90% of reference lengths, and subgenomic reads were subsampled from total viral reads with a custom script.
Reads were base called using the Guppy workflow (v5.0.16) with the configuration of rna_R9.4.1_70bps_hac.cfg or using the Dorado workflow (v0.3.4) with the configuration of rna002_70bps_hac@v3. The resulting FASTQ files were aligned to the SINV genome (GenBank:
4
Nanopore Sequencing of mRNA: Optimized Protocol
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Nanopore library followed the SQK-RNA002 kit (Oxford Nanopore) recommended protocol, the only modification was the input mRNA quantity increased from 500 to 1000 ng, all other consumables and parameters were standard. Final yields were evaluated using the Qubit HS dsDNA kit (Thermofisher Q32851) with minimum RNA preps reaching at least 150 ng. For all conditions, sequencing was performed on FLO-MIN106 flow cells either using a MinION MK1C or MinION sequencer running Minknow v20.06.5 and guppy v4.09. Basecalling was performed during the run using the fast-basecalling algorithm with a Q score cutoff >7. Long read alignment (to ME49-Toxodb-13 and TAIR10 reference fasta files) was performed using Minimap2 (ver 2.1) with the following parameters: ‘-ax splice -k14 -uf -G 5000 t 10 --secondary =no –sam-hit-only’ for Toxoplasma and ‘-ax splice -k14 -uf -G 20000 t 10 --secondary =no –sam-hit-only’ for Arabidopsis. Aligned reads were converted to bam, sorted, and indexed using Samtools. For T. gondii datasets, most sequencing runs were stopped after having generated between 400 k and 500 k of aligned reads to keep a standard of comparison (T. gondii reads varying between 30% and 70% of total mRNA depending on the preparation).
5
Nanopore Direct RNA Sequencing Protocol
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RNA was prepared for nanopore direct RNA sequencing following the Oxford Nanopore Technologies (ONT) SQK-RNA002 kit protocol, including the optional reverse transcription step recommended by ONT. RNA was sequenced in-house on the minION platform using ONT R9.4.1 flow cells. Total reads (in millions) passing default filters were Cytoplasm: 2.5, Nuclear: 1.8, PABPC IP in Cytoplasm: 1.9, PABPN IP in Nucleus: 0.9.
6
Direct RNA Nanopore Sequencing Protocol
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Libraries for direct RNA nanopore sequencing were prepared from poly(A)-tailed RNAs according to the SQK-RNA002 Kit protocol (Oxford Nanopore, Version: DRS_9080_v2_revS_14Aug2019) with minor modifications. Briefly, Agencourt AMPure XP magnetic beads (Beckman Coulter) in combination with 1 µL of RiboGuard RNase Inhibitor (Lucigen) were used instead of the recommended Agencourt RNAclean XP beads to purify samples after enzymatic reactions. Also, the RTA adapter was replaced by custom adapters described in https://github.com/Psy-Fer/deeplexicon to barcode the samples (Smith et al. 2020 (link)). RNA was linearized using Maxima RT H Minus enzyme (Thermo Fisher Scientific, #EP0751) and incubated for 30 min at 60°C followed by a heat inactivation step of 5 min at 85°C. After RT reactions, cDNA was quantified using the Qubit DNA HS Assay Kit (Thermo Fisher Scientific), and equimolar amounts of DNA were used for ligation of the RNA Adapter (RMX) in a single tube. Subsequent reactions were performed according to the protocols recommended by Oxford Nanopore Technologies. Finally, libraries were loaded onto R9.4 flow cells (Oxford Nanopore Technologies) and sequenced on a MinION device for 48 h. Direct RNA sequencing was performed for two biological replicates of wild-type and KsgA-deletion samples.
7
Nanopore Sequencing of Viral RNA
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Total cellular RNA was extracted from harvested infected cells using the TRIzolTM reagent (Cat. No. 15596026, Ambion, ThermoFisher, Horsham, UK) at 1 mL per 107 cells, washed twice with 70% ice-cold ethanol and stored at –80 °C under 70% ethanol until use. On the day of sequencing, RNA was resuspended in sterile water, enriched for polyadenylated RNA and sequenced as described previously using the SQK-RNA002 kit and MINI06D R9 version of the flow cells (Oxford Nanopore Technologies, Oxford, UK) according to the vendors’ protocols [32 (link)].
8
SINEUP-GFP RNA Nanopore Sequencing
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Libraries of the direct SINEUP-GFP RNA transcribed in vitro and in-cell were prepared with an SQK-RNA002 kit (Oxford Nanopore Technology). The original reverse-transcription adapter (RTA) was used for in vitro samples and four barcoded RTA adapters described by Leger et al.78 (link) were used for in-cell samples. The libraries were applied to MinION Mk1C sequencer (Oxford Nanopore Technologies) and sequenced for 72 h.
9
Nanopore Direct RNA Sequencing Protocol
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For ND, D5, and D7 samples, total RNA was extracted using an RNAprep Pure Kit (polysaccharides and polyphenolics-rich) (Code no. DP441, Tiangen Co. Ltd, Beijing, China) following the manufacturer's instructions and treated with DNase I to remove DNA. Libraries for DRS were prepared using the Oxford Nanopore Technologies SQK-RNA002 kit protocol. In brief, poly(A) + RNA was isolated using a Dynabeads mRNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA; 61006) and ligated to Nanopore RTA adapter; first-strand cDNA was synthesized using SuperScript III reverse transcriptase (Thermo Fisher; 18080093) and ligated to RNA adapter (RMX). A total of 75-μL library was loaded onto the flow cell (Gao et al., 2021) . Six libraries (two biological repeats for ND, N5, and N7) were constructed and run on a MinION flow cell (FLO-MIN106) independently. Details of DRS libraries and read statistics are provided in Supplemental Table S1. DRS raw data was analyzed using Guppy (v3.6.1) with default parameters to generate FASTQ, which was then converted to FASTA format and corrected using LoRDEC (Salmela and Rivals, 2014) with Illumina short reads (PRJNA315705; Li et al., 2019) .
10
RNA Extraction and Nanopore Sequencing
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RNA extraction and sequencing was as described previously [27 (link), 31 (link), 34 (link)]. Briefly, total RNA was extracted from the infected cell using TRIzol reagent (#15596026, Ambion) at 1ml of reagent per 107 cells, as per manufacturer’s recommendations except that the final wash of the RNA pellet in 70% ethanol was repeated a further two times (total of 3 washes). Total RNA was enriched for poly A tails using Dynabeads™ mRNA purification kit (#61006, Invitrogen) as per manufacturer’s instructions and we used 350ng of poly A enriched RNA per sequencing reaction. We used the SQK-RNA002 kits and MIN106D R9 version flow cells (Oxford Nanopore Technologies), following the manufacturer’s protocols.
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