For whole genome sequencing, a SMRTbell library with an insert size of 12,000 nt was prepared from the high molecular weight DNA template using SMRTbell Express Template Prep Kit 2.0 and sequenced on the PacBio Sequel system (Pacific Biosciences, Menlo Park, CA, USA). Sequencing was performed with the Sequel Binding Kit 2.0 using a 20-h movie collection time following the manufacturer’s protocol (Pacific Biosciences, Menlo Park, CA, USA). For transcriptome sequencing, Iso-seq libraries were prepared using the NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs, Ipswich, MA, USA), Iso-Seq Express Oligo Kit, and SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, Menlo Park, CA, USA). Iso-seq was performed as described above.
Nebnext single cell low input cdna synthesis and amplification module
Manufactured by New England Biolabs
Sourced in United States
The NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module is a kit designed for the synthesis and amplification of cDNA from small amounts of starting material, such as single cells or low-input RNA samples. The module includes reagents and protocols for reverse transcription and subsequent amplification of the resulting cDNA.
Automatically generated - may contain errors
Lab products found in correlation
Smrtbell express template prep kit 2, by Pacific Biosciences (6 mentions)
Iso seq express oligo kit, by Pacific Biosciences (3 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Pacbio sequel system, by Pacific Biosciences (1 mentions)
Sequel binding kit 2, by Pacific Biosciences (1 mentions)
Phanta high fidelity dna polymerase, by Vazyme (1 mentions)
Internal control 1, by Pacific Biosciences (1 mentions)
Pronex beads, by Promega (1 mentions)
Sequel 2 binding kit 2, by Pacific Biosciences (1 mentions)
7 protocols using nebnext single cell low input cdna synthesis and amplification module
1
Whole Genome and Transcriptome Sequencing
2
PacBio Iso-Seq Library Preparation
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PacBio DNA read and RNA Iso-Seq libraries were constructed by the KAUST Bioscience Core Lab using standard PacBio protocols. The cDNA synthesis was done using NEBNext Single Cell/Low input cDNA synthesis and amplification module (NEB, Cat No.: E6421S) with Iso-Seq Express Oligo Kit (Pacbio, 101-737-500) and Iso-seq library was built with SMRTbell Express Template Prep Kit 2.0 (PacBio, 100-938-900).
3
PacBio Iso-Seq library preparation
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The PacBio Iso-Seq full-length sequencing library was prepared using 300 ng of total RNA using NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (NEB, catalog no. E6421) and SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, catalog no. 100–938-900). This library was prepared in the Duke Center for Genomic and Computational Biology’s Sequencing Technologies Core facility, and was sequenced using three SMRT cells.
4
Single-cell IgG VDJ sequencing from oocytes
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RNA extraction and cDNA synthesis were performed with NEBNext® Single Cell/Low Input cDNA Synthesis and Amplification Module (Cat No.: E6421, NEB, Lpswich, MA, USA) according to the instruction from 100 oocytes. For IgG VDJ amplification, two round of PCR were done with two set of primers respectively. The primer sequences are in supplementary table 1. The DNA polymerase was Phanta high-fidelity DNA polymerase (Cat No.: P505-d1, Vazyme, Nanjing, Jiangsu, China). Then PCR products were ligated into cloning vector (Vazyme, Cat No.: C601-02) and transformed into competent DH5α, and 50 colonies (ligated cDNA from each colony corresponds to the mRNA of a single oocyte) were sent to GENEWIZ (Suzhou, Jiangsu, China) for sequencing.
5
PacBio Iso-Seq full-length RNA sequencing
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The PacBio Iso-Seq full-length sequencing library was prepared using 300 ng of total RNA using NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (NEB, catalog no. E6421) and SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, catalog no. 100-938-900). This library was prepared in the Duke Center for Genomic and Computational Biology’s Sequencing Technologies Core facility, and was sequenced using three SMRT cells.
6
Iso-Seq library preparation and sequencing
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First-strand cDNA synthesis and cDNA amplification were performed with a NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (New England Biolabs, MA, USA) and Iso-Seq Express Oligo Kit (Pacific Biosciences (PacBio), CA, USA) according to the procedure and checklist (i.e., Iso-Seq Express Template Preparation for Sequel and Sequel II Systems) provided by PacBio. Amplified cDNA samples were purified with ProNex beads (Promega, WI, USA) using the standard workflow and assuming that most transcripts are ~2 kb. Quantification was performed using a Qubit Fluorometer (Thermo Fisher Scientific) and size distribution was confirmed by a Femto Pulse System.
The Iso-Seq library was prepared by going through the steps of DNA damage repair, end repair and A-tailing, overhang adopter ligation, and cleanup using a SMRTbell Express Template Prep Kit 2.0 (PacBio). Sequencing primer was annealed and polymerase binding, complex cleanup, and sample loading were performed with a Sequel II binding kit 2.1 and internal control 1.0 (PacBio) following the manufacturer’s protocol, and sequencing was performed using the Sequel IIe system. Using one SMRTcell per sample, movie runtime was set to 24 hours for each SMRTcell.
The Iso-Seq library was prepared by going through the steps of DNA damage repair, end repair and A-tailing, overhang adopter ligation, and cleanup using a SMRTbell Express Template Prep Kit 2.0 (PacBio). Sequencing primer was annealed and polymerase binding, complex cleanup, and sample loading were performed with a Sequel II binding kit 2.1 and internal control 1.0 (PacBio) following the manufacturer’s protocol, and sequencing was performed using the Sequel IIe system. Using one SMRTcell per sample, movie runtime was set to 24 hours for each SMRTcell.
7
PacBio Iso-Seq full-length transcriptome
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300 ng total RNA was used to prepare each PacBio Iso-Seq full-length transcript sequencing library. Libraries were prepared in the Duke Center for Genomic and Computational Biology's Sequencing and Genomic Technologies Core Facility using the NEBNext Single Cell/Low Input cDNA Synthesis and Amplification Module (NEB, catalog no. E6421) and SMRTbell Express Template Prep Kit 2.0 (Pacific Biosciences, catalog no. 100-938-900). Each library was sequenced with three SMRT cells.
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