RNA isolation and library preparation is fully described in Butler, et al. (Butler et al., 2021 (link)). Briefly, library preparation on all the nasopharyngeal swab samples’ total nucleic acid (TNA) were treated with DNAse 1 (Zymo Research, Catalog # E1010). Post-DNAse digested samples were then put into the NEBNext rRNA depletion v2 (Human/Mouse/Rat), Ultra II Directional RNA (10 ng), and Unique Dual Index Primer Pairs were used following the vendor protocols from New England Biolabs. Kits were supplied from a single manufacturer lot. Completed libraries were quantified by Qubit or equivalent and run on a Bioanalyzer or equivalent for size determination. Libraries were pooled and sent to the WCM Genomics Core or HudsonAlpha for final quantification by Qubit fluorometer (ThermoFisher Scientific), TapeStation 2,200 (Agilent), and qRT-PCR using the Kapa Biosystems Illumina library quantification kit.
Unique dual index primer pairs
Manufactured by New England Biolabs
Unique Dual Index Primer Pairs are oligonucleotide sequences designed for use in next-generation sequencing applications. They allow the introduction of dual barcode sequences during library preparation to enable multiplexing of samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit fluorometer, by Thermo Fisher Scientific (4 mentions)
Nebnext rrna depletion v2 human mouse rat, by New England Biolabs (2 mentions)
Ultra 2 directional rna 10 ng, by New England Biolabs (2 mentions)
Dnase 1, by Zymo Research (2 mentions)
Tapestation 2200, by Agilent Technologies (2 mentions)
Hiseq 4000, by Illumina (2 mentions)
Novaseq 6000, by Illumina (2 mentions)
Dnase, by New England Biolabs (1 mentions)
Nebnext enzymatic methyl seq em seq kit, by New England Biolabs (1 mentions)
Nebnext ultra 2 fs, by New England Biolabs (1 mentions)
Nebnext ultra 2 fs kit, by New England Biolabs (1 mentions)
5 protocols using unique dual index primer pairs
1
RNA Isolation and Directional Library Prep
2
RNA Isolation and Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation and library preparation is fully described in Butler et al. (2021) (link). Briefly, library preparation on the all nasopharyngeal swab samples’ total nucleic acid (TNA) were treated with DNase 1 (Zymo Research, Catalog # E1010). Post-DNase digested samples were then put into the NEBNext rRNA depletion v2 (Human/Mouse/Rat), Ultra II Directional RNA (10 ng), and Unique Dual Index Primer Pairs were used following the vendor protocols from New England Biolabs. Kits were supplied from a single manufacturer lot. Completed libraries were quantified by Qubit or equivalent and run on a Bioanalyzer or equivalent for size determination. Libraries were pooled and sent to the WCM Genomics Core or HudsonAlpha for final quantification by Qubit fluorometer (ThermoFisher Scientific), TapeStation 2200 (Agilent), and qRT-PCR using the Kapa Biosystems Illumina library quantification kit.
3
Enzymatic Methyl-seq Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries were prepared with 1, 2, 5 or 10ng DNA using the NEBNext® Enzymatic Methyl-seq (EM-seq™) kit and Unique Dual Index Primer pairs (New England Biolabs) input according to manufacturer’s instructions (NEB #E7120 S/L v6.0_3/2). Unmethylated lambda phage DNA and plasmid pUC19 DNA, methylated at CpG sites, were added as negative and positive controls, respectively. A total of 12 amplification cycles were used for 1ng of input DNA and 10 cycles for other input amounts. Libraries were pooled equimolarly and then sent for sequencing.
4
High-throughput DNA library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 20 ng amplified cDNA or circular DNA was used for library preparation using the NEBNext Ultra II FS (New England Biolabs) according to the manufacturer’s protocol. Samples were barcoded using unique dual-index primer pairs (New England Biolabs) and libraries were pooled and sequenced on a HiSeq 4000 instrument (Illumina) or a NovaSeq 6000 instrument with 2× 150-bp paired-end reads for circular DNA libraries and 2× 75-bp paired-end reads for cDNA libraries.
5
Single-Cell Extrachromosomal Circular DNA Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TR14 scCircle-seq data were deposited in the European Genome-phenome Archive (EGA) under the accession number: EGAS00001007026. A detailed description of the single-cell extrachromosomal circular DNA and transcriptome sequencing (scEC&T-seq) protocol is available on Nature protocol exchange (DOI: 10.21203/rs.3.pex-2180/v1). In short, single cells were separated into individual wells of a 96-well plates using FACS. Separation of genomic DNA and mRNA was performed as described in the G&T-seq protocol by Macaulay et al. 201549 (link). Genomic DNA of single cells was purified using 0.8× AMPure XP beads and subjected to exonuclease digestion and rolling-circle amplification as described in Chamorro González et al, 2023 (in press). All single-cell libraries were prepared using the NEBNext Ultra II FS kit (New England Biolabs) following the manufacturer’s instructions but using one-fourth volumes. Unique dual index primer pairs (New England Biolabs) were used to barcode single-cell libraries. Pooled libraries were sequenced on the Illumina HiSeq 4000 or the NovaSeq 6000 platform with 2 × 150bp paired-end reads for genomic DNA and circular DNA libraries and 2 × 75 bp paired-end reads for cDNA libraries.
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!