Single-molecule real-time (SMRT) sequencing was performed to detect large deletions targeting the human CLCN5 gene. A region of 4862 bp was amplified by LongAmp® Hot Start Taq 2× Master Mix (New England Biolabs, catalog # M0533) with primers hCLCN5-F3 and hCLCN5-R3. DNA was submitted to the GCB Sequencing and Genomic Technologies Shared Resource (Duke University, Durham, NC) for SMRT sequencing (Sequel I). One SMRTcell was used for eight barcoded samples. Demultiplexed circular consensus (CCS) reads received from the sequencing center were used to search for large deletions by comparing with the reference sequence through R programming. Specifically, all sequences were searched for the presence of a 25-bp 5′ sequence and a 25-bp 3′ sequence (see Supplementary Table S2 for sequences), each allowing a 2-nt difference (92% identity) to accommodate possible sequencing errors. These two regions are at the 5′ and 3′ ends of the sequenced DNA. The distances between the two 25-bp regions were then calculated for each read. Reads with distances that differed from the reference sequence and without intact sgRNA target sites were regarded as reads with deletions. The counts and the sequences of the reads with deletions were listed for further analysis.
Longamp hot start taq 2 master mix
Manufactured by New England Biolabs
LongAmp Hot Start Taq 2× Master Mix is a premixed, ready-to-use solution for PCR amplification. It contains Taq DNA polymerase, dNTPs, and reaction buffer components.
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Lab products found in correlation
Monarch dna gel extraction kit, by New England Biolabs (1 mentions)
Sequel 2 smrt cell 8 m, by Pacific Biosciences (1 mentions)
Genomic tip 20 g kit, by Qiagen (1 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (1 mentions)
Spriselect, by Beckman Coulter (1 mentions)
Thermolabile exonuclease 1, by New England Biolabs (1 mentions)
5 protocols using longamp hot start taq 2 master mix
1
Detecting CLCN5 Gene Deletions via SMRT Sequencing
2
Quantifying MBNL1/MBNL2 Genome Editing
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To quantify the genome editing outcome of MBNL1/MBNL2, the intended genomic double-stranded break region was PCR-amplified using the LongAmp Hot Start Taq 2× Master Mix (NEB) according to the manufacturer’s protocol with primer deoxynucleotides pairs (IDT) flanking the expected mutation region. After DNA agarose gel electrophoresis, the PCR fragments were cut out and purified using the Monarch DNA Gel Extraction Kit (NEB). Indel analysis was performed using TIDE54 (link).
3
Comprehensive Long-Read Sequencing of SVs
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All 23 LRS-unique SVs were validated using long-range PCR followed by sequencing on a PacBio Sequel IIe system. Primers were designed using Primer3Input, and PCR was performed using NEB LongAmp Hot Start Taq 2 × Master Mix. For each PCR product, 500 ng was used as input for the library preparation and the normalized library was prepared according to the manufacturer’s instructions using the SMRTbell barcoded adapter complete prep kit. Finally, the library, with a loading concentration of 80 pm, was sequenced on a PacBio Sequel IIe system using a Sequel II SMRT Cell 8 M (PacBio, Menlo Park, CA, USA) with a movie time of 30 h and 0.7 h pre-extension time.
4
Quantifying Mitochondrial DNA Lesions
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gDNA was isolated from 1 × 106 cecum cells with the Qiagen Genomic-tip 20/G Kit using the Qiagen Genomic DNA buffer set applying the protocol for tissues. TE buffer (20×, 200 mM Tris⋅HCl, 20 mM EDTA, pH 8.0) was used in all steps. gDNA was quantified via Pico Green standard curve (Quant-iT PicoGreen dsDNA reagent; Invitrogen) (56 (link)) using Lambda (λ)/HindIII DNA (Thermo Fisher Scientific). Fluorescence was measured in a plate reader (20-s shaking, 485-nm excitation, 520-nm emission), and DNA quality was assessed with electrophoresis.
In total, 3.5 ng of gDNA was used for Long Amplicon PCR (56 (link), 57 (link)). PCR was performed in 25-µL reactions containing 1× LongAmp Hot Start Taq2× Master Mix (New England Biolabs), 0.1 µM primer, and nuclease-free water. Temperature profile was used as follows: 94 °C, 2 min; 94 °C, 15 s; x °C (individual), 12 min; 72 °C, 10 min; 4 °C, forever. Annealing temperatures and cycle numbers were adjusted for 8.7-kb β-globin fragment (accession number X14061) to 64 °C and 25 cycles; for 6.6-kb DNA-polymerase β (accession number AA79582) to 65 °C and 24 cycles; for 10-kb long mitochondrial fragment to 64 °C and 17 cycles; for 117-bp short mitochondrial fragment to 60 °C and 18 cycles. Ten microliters of PCR product were quantified in duplicate using Pico Green and lesion burden was calculated according to Furda et al. (56 (link)).
In total, 3.5 ng of gDNA was used for Long Amplicon PCR (56 (link), 57 (link)). PCR was performed in 25-µL reactions containing 1× LongAmp Hot Start Taq2× Master Mix (New England Biolabs), 0.1 µM primer, and nuclease-free water. Temperature profile was used as follows: 94 °C, 2 min; 94 °C, 15 s; x °C (individual), 12 min; 72 °C, 10 min; 4 °C, forever. Annealing temperatures and cycle numbers were adjusted for 8.7-kb β-globin fragment (accession number X14061) to 64 °C and 25 cycles; for 6.6-kb DNA-polymerase β (accession number AA79582) to 65 °C and 24 cycles; for 10-kb long mitochondrial fragment to 64 °C and 17 cycles; for 117-bp short mitochondrial fragment to 60 °C and 18 cycles. Ten microliters of PCR product were quantified in duplicate using Pico Green and lesion burden was calculated according to Furda et al. (56 (link)).
5
Targeted Long-Range PCR with UMI Tagging
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The first PCR reaction (PCR1) with two amplification cycles was used to target 5- to 6-kb region around the Cas9 cut site and simultaneously tag each template molecule with terminal UMIs using with a tailed primer pair (table S5). The first section of both tailed primers is a synthetic priming site used for downstream amplification, followed by the UMI and target specific sequences. The PCR1 reaction contained 500 ng of gDNA, 200 nM of each tailed primer in 100 μl of reaction (LongAmp Hot Start Taq 2× Master Mix, NEB). The PCR1 program consisted of initial denaturation (2 min at 94°C) and two cycles of denaturation (30 s at 94°C), annealing (30 s at 60°C), and extension (6 min at 65°C). After completion of PCR1, 5 μl of thermolabile exonuclease I (NEB, M0568) was added to the PCR1 reaction and incubated at 37°C for 4 min followed by heat inactivation at 80°C for 1 min to degrade all single-stranded DNA present in the PCR1 mixture. The PCR1 product was purified using SPRIselect (Beckman Coulter, B23317) and eluted in 30 μl of water.
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