Genomic DNA was bisulfite-modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) and the converted DNA was PCR-amplified using Taq™ Hot Start Version (TAKARA) in accordance with the provided guidelines. Primers used for PCR amplifications were forward 5′-TTTTTATTTTTAGGGAATATTTTTTGG-3′, reverse 5′- TACACACAATAAATCACACAAATCC-3’. The PCR products were electrophoresed in 1% agarose gels and purified using a TIANquick Midi purification kit (Tiangen Biotech) following the manufacturer's instructions. The purified products were then ligated into pMD-19T and transformed into DH5α competent cell. The successful construction of cloning plasmids was identified by colony PCR and ten positive clones for each subject were randomly selected for sequencing.
Taq hot start version
Manufactured by Takara Bio
Sourced in Japan, China
The Taq™ Hot Start Version is a DNA polymerase enzyme designed for the amplification of DNA sequences. It is heat-activated, providing enhanced specificity and sensitivity for PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator v1.1 cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Applied biosystems 3500xl capillary sequencer, by Thermo Fisher Scientific (2 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (2 mentions)
Rnaiso plus kit, by Takara Bio (2 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Tianquick midi purification kit, by Tiangen Biotech (1 mentions)
Lightcycler 480, by Roche (1 mentions)
12 protocols using taq hot start version
1
Bisulfite Conversion and DNA Methylation Analysis
2
Bisulfite Sequencing PCR Protocol
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TaKaRa Taq™ Hot Start Version (Tokyo, Japan) kit was applied for bisulfite sequencing PCR (BSP) reaction with primers also as reported 22 , 23 . BSP conditions was performed as our previous study 22 , 23 . BSP products cloning sequencing was performed as described previously 22 , 23 . Six independent clones from each specimen were sequenced (BGI Tech Solutions Co., Shanghai, China).
3
Variant Detection via PCR and Sanger
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Polymerase chain reaction amplification was performed using the Taq Hot Start version (TaKaRa, Japan) to eliminate false‐positive variants detected by next‐generation sequencing. Sanger sequencing was then performed for all putative pathogenic variants using the Big Dye v.1.1 terminator cycle sequencing kit and Applied Biosystems 3500xl capillary sequencer (Applied Biosystems, Foster City).
4
Poly(A) Tail Validation of mRNA
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To assess if the mRNA had the poly(A) tail as designed, the mRNAs were converted into cDNA using d(T) self-designed primer 5′-GCGACCACCGATTTTTTTTTTTT-3′ and the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA was PCR amplified using the TaKaRa Taq Hot Start Version (TaKaRa Bio, Kyoto, Japan, R007A) and the primers: 1: 5′-AATGATACGGCGACCACCGA-3′ and 2: 5′-AAGCACGCAGCAATGCAG CT-3′. Amplified DNA products were subjected to Sanger sequencing.
5
SLIT2 Promoter Methylation Analysis
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Bisulfite sequencing PCR (BSP) was further performed to detect SLIT2 promoter methylation using TaKaRa Taq™ Hot Start Version (Tokyo, Japan). The primers for SLIT2 promoter methylation detected by BSP were reported previously [17 (link)]. The details of BSP can be found in our previous study [17 (link)]. Six independent clones from each specimen were selected for Sanger sequencing (BGI, Shanghai, China).
6
RNA Extraction and Reverse Transcription
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Total RNA was extracted and reversely transcribed into cDNA according to the instructions of RNAiso Plus kit and PrimeScript ™ RT reagent Kit with gDNA Eraser (TaKaRa, China). The cDNA was amplified by PCR following the protocol of TaKaRa Taq ™ Hot Start Version (TaKaRa, China). The sequence of forward primer of BPTF was 5′-AATCGGAGAAGTCCAACGGG-3′; the sequence of reward primer of BPTF was 5′-TTGCCCTATGTGATGCCCAG-3′. They were produced by life Technologies Company (Invitrogen, Carlsbad, USA).
7
Reverse Transcription and PCR Amplification
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The total RNA isolated from 293T and A549 cells was reverse transcribed into cDNA, respectively, with the same procedures in the standard protocols. 60 μL of mixture was prepared with 1× PCR buffer (Mg2+ plus), 250 μM dNTPs, 500 nM forward and reverse primers each, 1 U TaKaRa Taq™ Hot Start Version and 12 μL reverse transcription product. The mixture was experienced 95 °C for 2 min to denature the hybridized RNA/DNA double strands and 40 cycles containing 95 °C for 20 s, 65 °C for 20 s and 72 °C for 30 s to complete the PCR amplification. The PCR products were sequenced by Sangon Biotech.
8
Poly(A) Tail Validation of mRNA
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To assess if the mRNA had the poly(A) tail as designed, the mRNAs were converted into cDNA using d(T) self-designed primer 5′-GCGACCACCGATTTTTTTTTTTT-3′ and the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific). cDNA was PCR amplified using the TaKaRa Taq Hot Start Version (TaKaRa Bio, Kyoto, Japan, R007A) and the primers: 1: 5′-AATGATACGGCGACCACCGA-3′ and 2: 5′-AAGCACGCAGCAATGCAG CT-3′. Amplified DNA products were subjected to Sanger sequencing.
9
Quantifying TRIM10 Methylation in BM Cells
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Genomic DNA Purification Kit (Vazyme, China) was used to extract genomic DNA from BM mononuclear cells. Then the CpGenome DNA Modification Kit (Vazyme, China) was applied to modify genomic DNA followed by storage at −80 °C. Takara Taq™ Hot Start Version (Tokyo, Japan) was applied to detect TRIM10 methylation using MSP with primers presented in Supplementary Table S2 . MSP reaction was performed on a LightCycler 480 real-time PCR instrument (Roche, Switzerland). The normalized ratio (NM-TRIM10) was applied to evaluate the level of TRIM10 methylation. NM-TRIM10 was calculated using the following formula: NM-TRIM10 = (EM-TRIM10)ΔCT M-TRIM10 (control-sample) ÷ (EALU) ΔCT ALU(control-sample).
10
Optimize Pathogenic Variant Detection
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PCR amplification was optimized in accordance with the manual for Taq™ Hot Start version (TaKaRa, Japan). To exclude any possible false-positive errors, Sanger sequencing was performed for all pathogenic or likely pathogenic variants using the Big Dye v.1.1 terminator cycle sequencing kit and an Applied Biosystems 3500xl capillary sequencer (Applied Biosystems, CA). The technically uncovered 3,972 bp regions of the 72 selected genes were sequenced directly by Sanger sequencing.
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