The sequencing libraries were prepared using CEL-Seq2 (Hashimshony et al., 2016 (link)). RNA from sorted cells was extracted using TRIzol reagent (ThermoFisher, 15596018). 10 ng RNA was used for first strand cDNA synthesis using barcoded primers (the specific primers for each sample were listed in Supplementary Table S9 ). The second strand was synthesized by NEBNext Second Strand Synthesis Module (NEB, E6111L). The pooled dsDNA was purified with AMPure XP beads (Beckman Coulter, A63880), and subjected to in vitro transcription (IVT) using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S), then treated with ExoSAP-IT (Affymetrix, 78200). IVT RNA was fragmented using RNA fragmentation reagents (Ambion), and underwent another reverse transcription step using random hexamer RT primer-5’-GCC TTG GCA CCC GAG AAT TCC ANN NNN N-3’ to incorporate the second adapter. The final library was amplified with indexed primers: RP1 and RPI1 (Supplementary Table S9 ), and the bead purified library was quantified with 4200 TapeStation (Agilent Technologies), and paired-end sequenced on Nextseq 500 V2 (Illumina), Read 1: 15 cycles; index 1: 6 cycles; Read 2: 60 cycles.
Nebnext second strand synthesis module
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Second Strand Synthesis Module is a laboratory kit that provides reagents and enzymes required for the synthesis of the second strand of complementary DNA (cDNA) from a single-stranded RNA template. It is designed to facilitate the conversion of RNA into double-stranded cDNA, which is a common step in various molecular biology workflows, such as gene expression analysis and library preparation for next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Miseq instrument, by Illumina (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (3 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (3 mentions)
Exosap it, by Thermo Fisher Scientific (3 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Tapestation 4200, by Agilent Technologies (2 mentions)
Nextera xt, by Illumina (2 mentions)
Xgen ngs rna, by Integrated DNA Technologies (2 mentions)
Mag bind total pure ngs, by Omega Bio-Tek (2 mentions)
Nextera xt, by Omega Bio-Tek (2 mentions)
Tapestation, by Agilent Technologies (1 mentions)
Nextseq 500 550 high output kit v2, by Illumina (1 mentions)
Purelink pcr purification kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Nextera xt dna kit, by Illumina (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Random hexameric primers, by Promega (1 mentions)
Tri reagent, by Molecular Research Center (1 mentions)
Revertaid reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
8 protocols using nebnext second strand synthesis module
1
Single-Cell Transcriptome Profiling by CEL-Seq2
2
CEL-Seq2 Library Preparation for Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequencing libraries were prepared using CEL-Seq2 (Hashimshony et al., 2016 (link)). RNA from sorted cells was extracted using TRIzol reagent (ThermoFisher, 15596018). Ten ng RNA was used for first strand cDNA synthesis using barcoded primers (the specific primers for each sample were listed in Supplementary file 1b ). The second strand was synthesized by NEBNext Second Strand Synthesis Module (NEB, E6111L). The pooled dsDNA was purified with AMPure XP beads (Beckman Coulter, A63880), and subjected to in vitro transcription (IVT) using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S), then treated with ExoSAP-IT (Affymetrix, 78200). IVT RNA was fragmented using RNA fragmentation reagents (Ambion) and underwent another reverse transcription step using random hexamer RT primer-5’-GCC TTG GCA CCC GAG AAT TCC ANN NNN N-3’ to incorporate the second adapter. The final library was amplified with indexed primers: RP1 and RPI1 (Supplementary file 1b ), and the bead purified library was quantified with 4,200 TapeStation (Agilent Technologies) and paired end sequenced on Nextseq 500 V2 (Illumina), Read 1: 15 cycles; index 1: 6 cycles; Read 2: 60 cycles.
3
Single-cell RNA-seq of NK and ILC Subsets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The sequencing library was prepared using CEL‐Seq2 (Hashimshony et al, 2016 (link)). RNA from sorted cells was extracted using TRIzol reagent (ThermoFisher, 15596018). Ten‐nanogram RNA was used for first‐strand cDNA synthesis using barcoded primers (the specific primers for each sample were listed in Dataset EV9 ). The second strand was synthesized by NEBNext Second Strand Synthesis Module (NEB, E6111L). The pooled dsDNA was purified with AMPure XP beads (Beckman Coulter, A63880), and subjected to in vitro transcription (IVT) using HiScribe T7 High Yield RNA Synthesis Kit (NEB, E2040S), then treated with ExoSAP‐IT (Affymetrix, 78200). IVT RNA was fragmented using RNA fragmentation reagents (Ambion) and underwent another reverse transcription step using random hexamer RT primer‐5′‐GCC TTG GCA CCC GAG AAT TCC ANN NNN N‐3′ to incorporate the second adapter. The final library was amplified with indexed primers: RP1 and RPI1 or RPI2 (as indicated in Dataset EV9 ), and the beads purified library was quantified with 4200 TapeStation (Agilent Technologies), and paired‐end sequenced on Nextseq 500 V2 (Illumina), Read 1: 15 cycles; index 1: 6 cycles; Read 2: 60 cycles. Libraries of CD56hi, CD56dim, CD56− NK cells, and ILCs from people who were HIV‐1−, or HIV‐1+ and viremic, under ART, or elite controllers, were sent to Novogene for sequencing by NovaSeq.
4
cDNA Preparation and Genomic Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA preparation from RNA for genomic sequencing started with the synthesis of the first and second cDNA strands using the SuperScriptTM VILOTM MasterMix kit (Thermo Fisher Scientific, Waltham, MA, USA) and NEBNext® Second Strand Synthesis Module (New England BioLabs, Ipswich, MA, USA), respectively. The cDNA purification reaction was performed using the PureLink® PCR Purification Kit (Thermo Fisher Scientific, Waltham, MA, USA). All steps followed the manufacturer’s recommendations for the respective kits. The genomic library was prepared following the instructions of the Nextera XT DNA kit (Illumina, San Diego, CA, USA). Quantification and fragmentation level assessment were performed using the DNA HS Kit Assay on the Qubit 4.0 instrument and the Agilent RNA 6000 Pico kit in the Bioanalyzer 2100 instrument, respectively. Upon confirming that the library had the desired quantity and size of fragments, sequencing was performed on the NextSeq 500 platform (Illumina) using the NextSeq 500/550 High Output Kit v2.5 (300 cycles) and paired-end methodology, as recommended by the manufacturer.
5
Illumina Library Prep from Total RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries from randomly primed total RNA were prepared using the xGen NGS RNA (Integrated DNA Technologies) kit for HEK293 and the Nextera XT (Illumina) kit for E. coli cells. cDNAs were converted to double stranded DNA (dsDNA) by NEBNext second-strand synthesis module (New England Biolabs) using 2 h incubation at 16 °C. dsDNA was purified and size selected using magnetic beads (Mag-Bind Total Pure NGS, Omega Bio-Tek) using a 0.65x bead ratio. Libraries were then generated following the Nextera XT manufacturer protocol, followed by purification and size-selection by magnetic beads (Mag-Bind Total Pure NGS, Omega Bio-Tek) using a 0.56x bead ratio. Libraries were sequenced on an Illumina MiSeq instrument using 2×300 paired-end sequencing (v3 chemistry).
6
NGS Library Preparation for HEK293 and E. coli
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Libraries from randomly primed total RNA were prepared using the xGen NGS RNA (Integrated DNA Technologies) kit for HEK293 and the Nextera XT (Illumina) kit for E. coli cells. cDNAs were converted to double stranded DNA (dsDNA) by NEBNext second-strand synthesis module (New England Biolabs) using a 2 h incubation at 16°C. dsDNA was purified and size selected using magnetic beads (Mag-Bind Total Pure NGS, Omega Bio-Tek) using a 0.65× bead ratio. Libraries were then generated following the Nextera XT manufacturer protocol, followed by purification and size-selection by magnetic beads (Mag-Bind Total Pure NGS, Omega Bio-Tek) using a 0.56× bead ratio. Libraries were sequenced on an Illumina MiSeq instrument using 2 × 300 paired-end sequencing (v3 chemistry) (Supplementary Table S4 ).
7
Orthobunyavirus RNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Orthobunyairuses were obtained from the Russian State Collection of Viruses in the form of lyophilized infected suckling mouse brains (Table 1 ). Total RNA was isolated from vials with 1 mL of TRI Reagent (Molecular Research Center, Cincinnati, OH, USA). Total RNA was additionally purified using the RNeasy MinElute Cleanup Kit (Qiagen, Hilden, Germany) followed by ribosomal RNA depletion using the GeneRead rRNA Depletion Kit (Qiagen) according to the manufacturers’ instructions. Purified RNA was reverse-transcribed with RevertAid Reverse Transcriptase (Thermo Fisher Scientific, Grand Island, NY, USA) using hexameric random primers (Promega, Madison, WI, USA). First strand cDNA was converted to double-stranded cDNA using the NEBNext Second Strand Synthesis Module (New England BioLabs, Ipswich, MA, USA) according to the manufacturer’s instructions. Resulting dsDNA was used to prepare next-generation sequencing libraries using the TruSeq DNA LT Library Prep Kit (Illumina, San Diego, CA, USA). A paired-end 250-bp protocol was used for sequencing indexed libraries on an Illumina MiSeq instrument.
8
TBEV Genome Sequencing with Illumina MiSeq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Amplicons previously used to sequence the TBEV-NL genome by Sanger sequencing (GenBank accession number LC171402) and RNA isolated from viral culture supernatants were sequenced using the Illumina MiSeq instrument (Illumina, San Diego, CA, USA). Viral RNA was transcribed into cDNA using the SuperScript III reverse transcriptase (Invitrogen) with random nonamers (New England Biolabs), followed by second strand DNA synthesis using NEBNext Second Strand Synthesis Module (New England Biolabs). dsDNA was concentrated using the DNA Clean & Concentrator™-5 kit (Zymo research) according to manufacturer’s instructions. Library preparation was performed using Nextera XT DNA library preparation kit (Illumina, San Diego, CA, USA), MiSeq PE300 sequencing (Illumina, San Diego, CA, USA) and demultiplexing of raw sequence data were all outsourced to BaseClear B.V. (Leiden, the Netherlands). The CLC Genomics workbench (CLCbio, Aarhus, Denmark) was used to trim the reads. Subsequently, all possible human reads were removed, remaining reads were mapped against the TBEV-EU reference genome (GenBank accession number NC_001672) and a consensus sequence was extracted.
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!