Genomic DNA was isolated from DG7710 cells by first treating with modified Wizard Genomic DNA Purification protocol for gram-positive bacteria (Promega): bacterial cell pellets were resuspended in 0.5 M EDTA, pH 8.0 supplemented with 200 μg ml−1 lysozyme (Sigma) and incubated at 37 °C for 25 min prior to pelleting and removing the supernatant. The standard Wizard protocol for gram-positives was then followed as described by the manufacturer (Promega). We used 200 ng (log phase) of plasmid as input for PCR of the CRISPR locus with Phusion DNA Polymerase (Thermo) with the following primer mix: oPN737 with oPN738, oPN757, oPN758, or oPN759 depending on whether JAV25, JAV25-BIM01, JAV25-BIM02, or JAV25-BIM03 was used. To differentiate between samples during multiplexed high-throughput sequencing, variants of oPN737 containing randomized 5 nucleotides (NNNNN) followed by 3-6 nucleotide barcode at 5’ end. Amplicons corresponding to the size of expanded CRISPR arrays were gel purified allowing for the removal of unexpanded CRISPR arrays. Purified amplicons were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina), using a final concentration of 1.36x Sample Purification Beads (Ilumina) following end repair for further size selection, followed by high-throughput sequencing with the MiSeq platform.
Trueseq nano dna library prep protocol
Manufactured by Illumina
The TruSeq Nano DNA Library Prep protocol is a laboratory equipment product designed for the preparation of DNA libraries. It provides a standardized workflow for the construction of DNA libraries from DNA samples, enabling subsequent sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Phusion dna polymerase, by Thermo Fisher Scientific (3 mentions)
Sample purification beads, by Illumina (2 mentions)
Qiaprep spin miniprep kit, by Qiagen (2 mentions)
Wizard genomic dna purification protocol, by Promega (1 mentions)
Lysozyme, by Merck Group (1 mentions)
Miseq sequencing, by Illumina (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Minelute pcr purification kit, by Qiagen (1 mentions)
4 protocols using trueseq nano dna library prep protocol
1
Genomic DNA Isolation and CRISPR Profiling
2
Sequencing CRISPR Array from Phage-infected Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmid DNA was extracted from liquid cultures after phage infection experiments (see protocols above) and used as a template for Phusion PCR to amplify the CRISPR array. After bands of the CRISPR array were extracted and purified from a 2% agarose gel, samples were prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina) and subject to Illumina MiSeq sequencing. Data analysis was performed in Python, similar to that described previously (33 (link)). In short, the spacer sequences were extracted and the number of reads for each spacer was recorded from the raw Illumina FASTQ files. For each unique spacer sequence matching the ФNM4γ4 genome, various characteristics were determined and recorded, including the spacer's frequency within the sequencing population (number of reads of that spacer compared to total spacer reads sequenced), the position of its protospacer in the ФNM4γ4 genome, the strand of the genome on which the protospacer is found, and the PAM sequence flanking the protospacer (see Supplementary File 1 ).
3
Plasmid Isolation and Spacer Library Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids were isolated from S. aureus pellets with a modified protocol of QIAprep Spin Miniprep Kit (Qiagen Cat. #27104), as previously described (Aviram et al., 2022 (link)). 250 ng of each sample were used as input for PCR, using the Phusion DNA Polymerase (Thermo Cat. #F530L) with primers NA162/NA163 for the spacer-library experiments, and with NA101/NA102 or NA169/170 for type III-A or type II-A naïve spacer acquisition, respectively (see Table S7 for a full list of primers used in this study).
Spacer library PCRs were analyzed by agarose gel electrophoresis, bands were excised and purified using QIAquick gel extraction kit (Qiagen Cat. #28704). Naïve spacer acquisition PCRs underwent cleanup with the MinElute PCR Purification Kit (Qiagen Cat. #28004) and size selection using PippnHT 3% cassette with a timed protocol set at extraction between 26 and 35 min. Size selected products were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina). For maintaining the small sized product, 2.2× Sample Purification Beads (Ilumina) were used after end repair. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.
Spacer library PCRs were analyzed by agarose gel electrophoresis, bands were excised and purified using QIAquick gel extraction kit (Qiagen Cat. #28704). Naïve spacer acquisition PCRs underwent cleanup with the MinElute PCR Purification Kit (Qiagen Cat. #28004) and size selection using PippnHT 3% cassette with a timed protocol set at extraction between 26 and 35 min. Size selected products were then prepared for sequencing with the TrueSeq Nano DNA Library Prep protocol (Illumina). For maintaining the small sized product, 2.2× Sample Purification Beads (Ilumina) were used after end repair. Illumina libraries underwent high-throughput sequencing with the MiSeq platform.
4
CRISPR Array Expansion Sequencing
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!