Oligonucleotides were obtained from TsingKe (Beijing, China). DNase I, restriction endonucleases, E. coli inorganic pyrophosphatase, E. coli Poly(A) Polymerase, RNA 5′ Pyrophosphohydrolase (RppH), T4 DNA ligase, NTPs, dNTPs, and RNA purification kits were from New England BioLabs (Ipswich, MA, United States). RNase inhibitor was from Thermo Fisher Scientific (Waltham, MA, United States). PrimeSTAR Max DNA Polymerase and Premix Taq DNA Polymerase, SMARTScribe and ProtoScript II Reverse Transcriptase are were from TAKARA (Shiga, Japan). DNA purification kit was from Axygen (Union City, CA, United States). Ni-NTA resin was from Qiagen (Hilden, Germany). Preparative Superdex S200 for gel filtration was from GE Healthcare (Chicago, IL, United States). Radiolabeled nucleotides were from PerkinElmer (Waltham, MA, United States). 2′-F-dNTPs were from TriLink (San Diego, CA, United States).
Smartscribe
Manufactured by Takara Bio
Sourced in Japan, United States
SMARTScribe is a high-performance reverse transcriptase enzyme designed for sensitive and efficient cDNA synthesis. It is optimized for use in a variety of RNA analysis applications, including gene expression profiling, RT-qPCR, and NGS library preparation.
Automatically generated - may contain errors
Lab products found in correlation
Ribozero rrna depletion kit, by Illumina (10 mentions)
Hiseq 2500, by Illumina (4 mentions)
Nextseq, by Illumina (3 mentions)
Hiseq 2500 sequencer, by Illumina (3 mentions)
Superscript 2, by Thermo Fisher Scientific (3 mentions)
Nextseq 2500, by Illumina (3 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Superscript 3, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Takara Bio (2 mentions)
Dntp mix, by New England Biolabs (2 mentions)
Rnasin plus, by Promega (2 mentions)
Smart seq2 lysis buffer, by New England Biolabs (2 mentions)
Oligo dt30vn primer, by New England Biolabs (2 mentions)
Triton x 100 10, by Merck Group (2 mentions)
Smart first strand buffer, by Takara Bio (2 mentions)
Nextseq 500 sequencer, by Illumina (2 mentions)
Rnase inhibitor, by Promega (2 mentions)
Primestar max dna polymerase, by Takara Bio (1 mentions)
35 protocols using smartscribe
1
High-throughput RNA and DNA Manipulation
2
High-Throughput Ribozyme Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ribozyme activity was determined as previously described (Bendixsen et al., 2021 (link)). Briefly, DNA templates were synthesized with the promoter for T7 RNA polymerase to enable in vitro transcription. Templates were synthesized with mixtures of phosphoramidites at variable positions. For the comprehensive double-mutant data set, templates were synthesized with 97% wild-type nucleotides and 1% each of the other three nucleotides. For the phylogenetic derived data set, the template was synthesized with an equal mixture of the naturally occurring nucleotides that were found at 13 positions that varied across 99 mammalian genomes. During in vitro transcription, RNA molecules self-cleaved at different rates. The reaction was stopped at 30 min, and the RNA was concentrated and reverse transcribed with a 5′-RACE protocol that appends a new primer site to the cDNA of both cleaved and uncleaved RNA (SMARTScribe, Takara). The cDNA was PCR amplified with primers that add the adaptors for Illumina sequencing. This procedure was done in triplicate with unique dual-indexes for each replicate. DNA was combined equimolar and sent for sequencing (GC3F, University of Oregon.) Sequencing was performed on a single lane of a HiSeq 4,000 using paired-end 150 reads.
3
Illumina cDNA Library Construction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Illumina cDNA libraries were generated using a modified version of the RNAtag-seq protocol57 (link). Briefly, 500 ng of total RNA was fragmented, depleted of genomic DNA, dephosphorylated, and ligated to DNA adapters carrying 5’-AN8−3’ barcodes of known sequence with a 5’ phosphate and a 3’ blocking group. Barcoded RNAs were pooled and depleted of rRNA using the RiboZero rRNA depletion kit (Epicentre). Pools of barcoded RNAs were converted to Illumina cDNA libraries in 2 main steps: (i) SMARTScribe (Takara Bio) reverse transcription of the RNA using a primer binding to the constant region of the barcoded adapter and addition of an adapter to the 3’ end of the cDNA by template switching58 (link); and (ii) PCR amplification using primers whose 5’ ends target the constant regions of the 3’ or 5’ adapters of the cDNA and whose 3’ ends contain the full Illumina P5 or P7 sequences. cDNA libraries were sequenced on an Illumina NextSeq for monoculture transcriptomic data and on the Illumina HiSeq 2500 platform to generate paired-end reads for metatranscriptomic data.
4
Sanger Sequencing of Viral cDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was isolated from 100 μl of virus-containing supernatant using the Quick-RNA MiniPrep kit (Zymo Research, R1031 and R1033). Five microliters of total RNA per reverse transcription reaction mixture and random hexamer primers were used with SMART-Scribe reverse transcriptase (TaKaRa Bio, ST0065) to generate cDNA. PCR was done on the generated cDNA using VSV- or transgene-specific primers. PCR products were electrophoresed on 1% agarose gels containing ethidium bromide in TBE buffer, and PCR products were cut from the agarose gel from which DNA was extracted following the DNA extraction kit protocol (Qiagen, 28706). In a microcentrifuge tube, Following the Eurofins Genomics instructions, DNA with a concentration between 20 and 60 ng/μl was combined with a single primer. The DNA and primer combinations were sent to Eurofins Genomics for Sanger sequencing. As per the Eurofins Genomics sequencing algorithm, any base pair that obtained a Phred quality score of 20 or lower was marked as nonspecific (N). A Phred quality score of 20 or lower indicates a base call accuracy between 90 and 99%. All sequencing results were analyzed with SnapGene 4.3 software.
5
Illumina cDNA Library Construction Protocol
6
Single-Cell RNA, TCR, and BCR Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA isolation was conducted with standard TRIzol protocol (Invitrogen, USA). Bulk TCR and BCR libraries were constructed by template switch and nested PCR-based protocol. Briefly, total RNA was reverse transcribed with SMARTscribe (Takara, Japan) following the manufacturer's instructions using C-region specific primer corresponding to TRAC and TRBC. 2 μL out of 10 μL solved cDNA was applied as the template for the 1st nested PCR KAPA Hot Start Ready Mix (KAPA, USA) following the manufacturer's instructions. 2nd and 3rd nested PCR were conducted with Phanta Max high fidelity polymerase (Vazyme, China), while the Illumina adapters were added to the fragments in the 3rd nested PCR. Beads-based purification was conducted after the 1st and the 2nd nested PCR and the final library was purified with cycle pure kit (Omega, USA). Single-cell mRNA, TCR, BCR libraries were constructed on 10X platform following the manufacturer's instructions. The DNA library was sequenced on Nova 2000 platform (Illumina, USA) under PE150 protocol.
7
RNA-seq Library Construction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA-seq library construction was based on Hunt’s protocol (Hunt, 2015 (link)) with modifications. Briefly, mRNA was enriched with Oligo d(T)25 Magnetic Beads (New England Biolabs), fragmented at 94°C for 3 min in 5× first strand buffer (Invitrogen), and reversely transcribed into cDNA by SMARTScribe (TaKaRa). Sample specific barcodes were incorporated into Illumina sequencing primers during PCR amplification by Phire II (Invitrogen), followed by 300–500 bp size selection using agarose gel electrophoresis, and purified by Zymoclean gel DNA recovery kit (Zymo Research). Library quality was assessed by High Sensitivity DNA Chips in the Agilent Bioanalyzer 2100. Qualified cDNA libraries were sequenced using the paired-end methods in the Illumina HiSeq 2500 platform at the College of Environment and Ecology, Xiamen University.
8
Single-Cell Transcriptomic Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single cells from tissue were manually picked under fluorescent microscopy using mouth pipette. Each of the harvested single cells was transferred into 2 μl of cell lysis buffer (CLB) in 0.2 ml PCR tubes. Libraries of isolated single cells were then prepared as per Smart-seq2 protocol [43 (link)] with modifications on reverse transcription and amplification cycles. Briefly, oligo-dT primed reverse transcription was performed with Smartscribe (Takara, Japan) reverse transcriptase and locked TSO oligonucleotide (Exiqon, Denmark) upon single cells. Full-length cDNA amplification was conducted by 20 cycles of PCR amplification with HiFi-HotStart ReadyMix (KAPA Biosystems, USA) and subsequent 0.6× AMPure beads purification (BD, USA). Barcoded libraries were fragmented and tagmented with Nextera XT Library Prep kit (Illumina). Pooled libraries with unique N5-N7 barcodes were sequenced using a Hiseq 2500 sequencer (Illumina) and single-end 50 bp reads flow cell.
9
Single-cell cDNA synthesis and amplification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reverse transcription (RT) and PCR steps were performed as previously described66 (link). Briefly, SMARTScribe (Takara, 639537) retrotranscriptase, RNAse inhibitor (Takara, 2313A) and a template-switching oligo were added to the cell lysate to perform the retrotranscription step. Immediately after, a PCR mix comprised of SeqAMP (Takara, 638509) and ISPCR primer (binding to a common adapter sequence in all cDNA molecules) was used for the PCR step with 24 cycles of amplification. Target-specific primers spanning patient-specific mutations were also added to RT and PCR steps (Supplementary Table 6a ). After cDNA synthesis, cDNA from up to 384 single-cell libraries was pooled, purified using Ampure XP Beads (0.6:1 beads to cDNA ratio; Beckman Coulter) and resuspended in a final volume of 50 μl of EB buffer (Qiagen). The quality of cDNA traces was checked using a high-sensitivity DNA kit in a Bioanalyzer instrument (Agilent Technologies).
10
DNA and RNA Extraction and Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted using a modified protocol from Edwards et al., 1991 (link). Instead of vacuum drying, the pelleted DNA was air-dried. Amplicons were directly sequenced. RNA was extracted from ≈100 mg wet weight tissue using Rneasy kit from Qiagen following the manufacturer’s instructions, including an on-column Dnase I digestion. The same amount of total RNA was used for complementary DNA (cDNA) synthesis using either Superscript II (Invitrogen) (SZ lab) or Bioscript (Bioline) (JLB lab) reverse transcriptase according to the manufacturer’s instructions with oligo-dT15 primer. 5’ and 3’ RACE-ready cDNA synthesis was performed using SMARTScribe reverse transcriptase (Takara) according to the manufacturer’s instructions but with the addition of random primer mix (NEB) to allow reverse transcription of long mRNA templates. See Supplementary file 1 , Table 1a for primer sequences.
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!