TCR clonotypes of the T cells were analyzed by next‐generation sequencing, as described previously.16 Briefly, total RNA was extracted from tumors using the AllPrep DNA/RNA mini kit (Qiagen), or RNA from sorted CD8+/Dextramer+ T cells was extracted using the PicoPure RNA Isolation Kit (Life Technologies) according to the manufacturer's instructions. cDNAs with common 5′‐RACE adapters were synthesized from total RNA using the SMART library construction kit (Clontech Laboratories). TCRα and TCRβ cDNAs were amplified by PCR using a forward primer for the SMART adapter and reverse primers corresponding to the constant regions of each TCRα and TCRβ chain. Illumina index sequences with barcodes were added using the Nextera XT Index kit (Illumina), then the library was sequenced using 300‐bp paired‐end reads on an Illumina MiSeq system using the MiSeq Reagent v3 600‐cycles kit (Illumina, USA). Tcrip software was used to analyze the sequence data.
Smart library construction kit
Manufactured by Takara Bio
Sourced in United States
The SMART library construction kit is a laboratory equipment product offered by Takara Bio. The kit is designed for the construction of cDNA libraries. It provides the necessary components and protocols to synthesize and amplify full-length cDNA from small amounts of input RNA.
Automatically generated - may contain errors
Lab products found in correlation
Miseq reagent v3 600 cycles kit, by Illumina (4 mentions)
Nextera xt index kit, by Illumina (3 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Allprep dna rna mini kit, by Qiagen (2 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (2 mentions)
Miseq system, by Illumina (1 mentions)
Nextera index kit, by Illumina (1 mentions)
Abioanalyzer, by Agilent Technologies (1 mentions)
Nextra xt index kit, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Tape station bioanalyzer, by Agilent Technologies (1 mentions)
7 protocols using smart library construction kit
1
TCR Sequencing of Tumor-Infiltrating T Cells
2
TCR Sequencing of ELISPOT-Positive Cells
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We performed TCR sequencing using the previously described methods [19 (link),23 (link)]. In brief, we extracted total RNA from the ELISPOT-positive or peptide-HLA-tetramer-positive cells. We synthesized cDNAs with a common 5′-RACE adapter from the total RNA using a SMART library construction kit (Clontech, Mountain View, CA, USA). We amplified the TCRα and TCRβ cDNAs by PCR using a forward primer for the SMART adapter and reverse primers corresponding to the constant region of each of the TCRα and TCRβ sequences. After adding the Illumina index sequences with a barcode using the Nextera XT Index Kit (Illumina, San Diego, CA, USA), we sequenced the prepared libraries by 300-bp paired-end reads on the Illumina MiSeq platform using a MiSeq Reagent v3 600-cycles kit (Illumina, San Diego, CA, USA). We analyzed the obtained sequence reads using Tcrip software [23 (link)].
3
Sequencing T Cell Receptor Repertoire
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We determined TCR sequences using the method we previously developed [35 (link), 49 (link)]. In brief, we extracted total RNAs from FACS-sorted dextramer-positive T cells. cDNAs with common 5′-RACE adapter were synthesized using SMART library construction kit (Clontech, Mountain View, CA). The fusion PCR was performed to amplify TCRA and TCRB cDNAs using a forward primer corresponding to the SMART adapter sequence and reverse primers corresponding to the constant region of each of TCRA and TCRB. After adding the Illumina index sequences with barcode using the Nextera Index kit (Illumina, San Diego, CA), the prepared libraries were sequenced by 300-bp paired-end reads on the MiSeq (Illumina). Obtained sequence reads were analyzed using Tcrip software [49 (link)].
4
Salivary Gland Transcriptome Analysis
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Total RNA was isolated from 300 pairs of salivary glands using TRI reagent following the
protocol provided by the manufacturer. RNA quality and integrity were assessed using a
Bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA libraries were constructed using
a “SMART” library construction kit from Clontech (Palo Alto, CA) as described by Chen et al. (2004) (link). Briefly, cDNA inserts were
ligated into the pPCRXL-TOPO plasmid contained in a TOPO TA cloning kit (Invitrogen,
Carlsbad, CA) instead of a phage vector. Individual clones were picked up for plasmid DNA
isolation, which were sequenced with the M13 forward and reverse primers following the
Sanger DNA sequencing method via a commercial contract (GENEWIZ, South Plainfield,
NJ).
protocol provided by the manufacturer. RNA quality and integrity were assessed using a
Bioanalyzer (Agilent Technologies, Santa Clara, CA). cDNA libraries were constructed using
a “SMART” library construction kit from Clontech (Palo Alto, CA) as described by Chen et al. (2004) (link). Briefly, cDNA inserts were
ligated into the pPCRXL-TOPO plasmid contained in a TOPO TA cloning kit (Invitrogen,
Carlsbad, CA) instead of a phage vector. Individual clones were picked up for plasmid DNA
isolation, which were sequenced with the M13 forward and reverse primers following the
Sanger DNA sequencing method via a commercial contract (GENEWIZ, South Plainfield,
NJ).
5
TCR Sequencing of Tumor and T Cells
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TCR sequencing was performed using the methods described previously.31 ,43 ,45 In brief, total RNAs were extracted from tumors with AllPrep DNA/RNA mini kit (Qiagen, Catalog number 80207) or sorted CD8+/Dextramer+ T cells with PicoPure RNA Isolation Kit (Life Technologies, Catalog number KIT0204). The cDNAs with common 5ʹ-RACE adapters were synthesized from total RNA using SMART library construction kit (Clontech, Catalog number 634901). The TCRA and TCRB cDNAs were amplified by PCR using a forward primer for the SMART adapter and reverse primers corresponding to the constant regions of each of TCRA and TCRB. After adding the Illumina index sequences with barcodes using the Nextera XT Index kit (Illumina, Catalog number FC-131-2004), the prepared libraries were sequenced by 300-bp paired-end reads on Illumina MiSeq platform, using MiSeq Reagent v3 600-cycles kit (Illumina, Catalog number MS-102-3003). Obtained sequence reads were analyzed using Tcrip software.31
6
High-Throughput TCR Sequencing Protocol
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TCR sequencing was performed using the methods described previously [20 (link), 27 (link), 28 (link)]. In brief, we extracted total RNAs from 2 × 105 T cells or tumor tissues, and cDNA was synthesized from total RNA with a 5’-Race adapter using the SMART library construction kit (Clontech). The TCRα and TCRβ cDNAs were amplified by PCR using a forward primer for the SMART adapter and a reverse primer corresponding to the constant region of TCRα and TCRβ. After adding the Illumina index sequences with barcode using the Nextra XT Index kit (Illumina), the prepared libraries were sequenced by 300-bp paired-end reads on the Illumina MiSeq platform, using MiSeq Reagent v3 600-cycles kit (Illumina). Obtained sequences were analyzed using Tcrip software.
7
Salivary Gland RNA Extraction and Sequencing
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Each RNA sample was extracted from a pool of 300 pairs of salivary glands from a species. Total RNA was extracted using TRI reagent following the protocol provided by the manufacturer. RNA quality and integrity were analyzed using a TapeStation Bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA samples were then reverse-transcribed to cDNAs. The cDNA samples were amplified using a ‘SMART’ library construction kit from Clontech (Palo Alto, CA) as described by Chen et al. (2004) (link). Briefly, amplified cDNA inserts were ligated into the pPCRXL-TOPO plasmid contained in a TOPO TA cloning kit (Invitrogen, Carlsbad, CA). The ligated plasmids were then transformed into individual bacteria. Bacterial clones were picked up individually for plasmid DNA isolation, which were sequenced with the M13 forward and reverse primers via a commercial contract (GENEWIZ, South Plainfield, NJ).
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