TALEN target sites as well as putative off-target sites were amplified from 100 ng genomic DNA by standard PCR using Q5® Hot Start High-Fidelity DNA Polymerase (NEB). PCR fragments were purified using either the QIAquick PCR Purification Kit or the QIAquick Gel Extraction Kit (both QIAGEN). Purified PCR products were pooled per sample and NGS libraries constructed using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (NEB). The resulting NGS libraries were quantified by ddPCR using the ddPCR™ Library Quantification Kit for Illumina TruSeq (Bio-Rad) and sequenced on Illumina HiSeq or NovaSeq platforms with 2 × 150 bp read length by NGS service provider Genewiz (part of Azenta Life Sciences). Reads were analyzed using the CRISPResso2 package (Clement et al., 2019 (link)) and the p values obtained from CRISPResso Compare. All primers used for Amplicon-NGS are listed in Supplementary Tables S11 .
Ddpcr library quantification kit for truseq
Manufactured by Illumina
The DdPCR™ Library Quantification Kit for Illumina TruSeq is a laboratory equipment product that enables precise quantification of DNA libraries prior to sequencing on Illumina platforms. The kit utilizes Droplet Digital PCR (ddPCR) technology to provide an accurate and sensitive method for determining library concentration.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy micro kit, by Qiagen (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ovation solo rna sequencing rna seq system, by Tecan (2 mentions)
Qx200 droplet digital pcr system, by Bio-Rad (2 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Qiaquick pcr purification kit, by Qiagen (1 mentions)
Novaseq, by Illumina (1 mentions)
Q5 hot start high fidelity dna polymerase, by New England Biolabs (1 mentions)
Nebnext ultra 2 dna library prep kit, by New England Biolabs (1 mentions)
Miseq reagent kit v2, by Illumina (1 mentions)
Miseq platform, by Illumina (1 mentions)
Miseq reagent kit v3, by Illumina (1 mentions)
6 protocols using ddpcr library quantification kit for truseq
1
TALEN off-target assessment by NGS
2
Benchmarking Nuclease Off-Target Effects
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The top 10 most highly predicted loci for off-target nuclease activity of Cas9-3 and Cas12a-1 were identified by Benchling online software, while TALEN sites were predicted using PROGNOS (http://bao.rice.edu/cgi-bin/prognos/prognos.cgi ) (Fine et al., 2014 (link)). PCR amplicons were designed to generate 150-200bp with the expected cut site in the centre. DNA from male healthy donor T cells edited with each nuclease platform (alongside untreated controls) was extracted at day 3 post nucleofection (Qiagen) and used as a template for on-target and off-target PCR reactions. The amplicons were then prepared for Illumina next generation sequencing by performing end repair, adapter ligation and bar coding using the NEBNext® UltraTM II DNA Library Prep Kit (NEB) according to the manufacturer’s instructions. Libraries were quantified using the ddPCR™ Library Quantification Kit for Illumina TruSeq (Biorad), before sequencing using MiSeq Reagent Kit v2, 500cycles on an Illumina MiSeq platform (Illumina). The generated paired-end reads were processed using the command line version of the CRISPResso2 pipeline (Clement et al., 2019 (link)), obtained editing frequencies were compared to the untreated control samples using a one-sided Fisher’s exact test. *, **, and *** indicate p < 0.05, p < 0.01, p < 0.001, respectively.
3
Illumina Paired-End Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Illumina paired-end sequencing libraries were prepared using the Accel-NGS 2S DNA Library kit for Illumina Platforms (Swift Biosciences), following manufacturer protocol version 2.0. Each individual DNA pool was given a unique multiplex identifying adapter (MID). Quality and concentration evaluations for amplicon pools, pre- and post-adapter addition, were visualized using a DNA 1000 Assay chip on a BioAnalyzer 2100. Library quality was assessed using the ddPCR Library Quantification Kit for Illumina TruSeq (Bio-Rad), according to manufacturer specifications, on the ddPCR platform. Prepared libraries were sequenced on an Illumina MiSeq platform at the Institute for Genome Sciences Genomics Resource Center at the University of Maryland School of Medicine. The libraries were sequenced in a paired-end manner using the MiSeq Reagent Kit v3 (Illumina, Inc.), generating paired-end 300-bp read lengths.
4
Droplet Digital PCR Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Similarly, for ddPCR titration, we used a series of dilutions made from the initial samples (1 × 10−6; 1 × 10−7; 1 × 10−8). Then, according to the kit instructions (ddPCR™ Library Quantification Kit for Illumina TruSeq, BioRad), we prepared mixes of 20 μl reactions per sample as follows: 10 μl of 2x ddPCR Supermix; 1 μl of 20x ddPCR library reaction assay; 5 μl DNAse-free water completed with 4 μl of the diluted sample to reduce pipetting errors. Droplets were generated, transferred to a thermocycler and submitted to the following cycling program: 95 °C heat activation for 10 min followed by 40 cycles of 30 seconds at 95 °C for denaturation and 1 min at 60 °C for annealing-extension steps. Finally, the PCR reaction was stopped with a last step at 98 °C for 10 min. The plate was then transferred and loaded into the ddPCR QX100 reader. Results obtained correspond to the number of molecules in one reaction. After dilution corrections, the number of molecules was converted into molarity. All samples were run in duplicate in three separate assays for a total of 18 values per libraries.
5
RNA-Seq Library Preparation from Small Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After NG2 sorting, cells were pelleted and RNA was extracted using RNeasy Micro Kit (QIAGEN) according to manufacturer's instructions. RNA integrity was assessed by RNA integrity number (RIN) values as shown in Supplementary Table 1 . NGS library prep was performed with NuGen Ovation SoLo RNA-Sequencing (RNA-seq) System following NuGen's standard protocol (M01406v2). For the first experiment (at P9) libraries were prepared with a starting amount of 1 ng and amplified in 14 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the ddPCR Library Quantification Kit for Illumina TruSeq in a QX200 Droplet Digital PCR system (BioRad). Details information on the NGS protocol are described in the SI.
6
RNA-seq analysis of NG2 sorted cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
After NG2 sorting, cells were pelleted and RNA was extracted using RNeasy Micro Kit (QIAGEN) according to manufacturer's instructions. NGS library prep was performed with NuGen Ovation SoLo RNA-Sequencing (RNA-seq) System following NuGen`s standard protocol (M01406v2). For the first experiment (at P9) libraries were prepared with a starting amount of 1 ng and amplified in 14 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the ddPCR Library Quantification Kit for Illumina TruSeq in a QX200 Droplet Digital PCR system (BioRad). Details information on the NGS protocol are described in the SI.
After NG2 sorting, cells were pelleted and RNA was extracted using RNeasy Micro Kit (QIAGEN) according to manufacturer's instructions. NGS library prep was performed with NuGen Ovation SoLo RNA-Sequencing (RNA-seq) System following NuGen`s standard protocol (M01406v2). For the first experiment (at P9) libraries were prepared with a starting amount of 1 ng and amplified in 14 PCR cycles. Libraries were profiled in a High Sensitivity DNA on a 2100 Bioanalyzer (Agilent technologies) and quantified using the ddPCR Library Quantification Kit for Illumina TruSeq in a QX200 Droplet Digital PCR system (BioRad). Details information on the NGS protocol are described in the SI.
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!