Lysed phage DNA from each sample was amplified and readied for Illumina deep sequencing by performing two rounds of PCR, as previously described (Garrett et al., 2020 ). Each PCR reaction was performed using Q5 High-Fidelity 2X Master Mix. For the first round of PCR, 10uL of lysed phage was used as the template in a 25 uL reaction. For the second round of PCR, 2 uL of the round 1 PCR product was then used as the template in a 50 uL reaction, with primers that add dual indexing sequences on either side of the insert. PCR products were then cleaned using AMPure XP beads and eluted in 50 uL water. DNA concentrations were quantified via Quant-iT PicoGreen dsDNA Assay Kit. Equimolar amounts of DNA from the samples, along with 10X the amount of the input library samples, was pooled, gel purified, and the final library was quantified using the KAPA Library Quantification Kit. Pools were sequenced on an Illumina MiSeq with 1×125 bp single end reads using a custom sequencing primer.
Deep sequencing
Manufactured by Illumina
Sourced in China
Deep sequencing is a high-throughput DNA sequencing technology that can rapidly and accurately determine the nucleotide sequence of large segments of genetic material. It is capable of generating millions of sequence reads in a single run, providing a comprehensive view of genetic information.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (2 mentions)
Nanoquant plate, by Tecan (2 mentions)
Truseq, by Illumina (1 mentions)
Plx sgrna, by Addgene (1 mentions)
Gibson assembly, by New England Biolabs (1 mentions)
Amplitaq gold 360 dna polymerase, by Thermo Fisher Scientific (1 mentions)
Miseq platform, by Illumina (1 mentions)
Prism 6, by GraphPad (1 mentions)
Total rna kit, by Qiagen (1 mentions)
Sybr green mix, by Yeasen (1 mentions)
Rnase free dnase 1, by New England Biolabs (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Fluorescence activated cell sorting (facs), by BD (1 mentions)
Amperase ung, by Thermo Fisher Scientific (1 mentions)
Oligotex mrna midi kit, by Qiagen (1 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hipure tissue dna mini kit, by Magen Biotechnology Co (1 mentions)
32 protocols using deep sequencing
1
Illumina Sequencing of Phage DNA
2
CRAC Experiment and Sequence Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The CRAC experiment and its analyses were performed as previously described58 –61 . Briefly, optimally growing cells carrying appropriate variations of HTP-tagged Xrn1 or Tail were UV crosslinked and harvested. RNA-protein complexes were captured tandemly on IgG sepharose and Ni-NTA followed by partial RNase digestion using RNace-IT. After ligation of sequencing adaptors, cDNA libraries were prepared by reverse transcription and PCR amplification followed by Illumina deep sequencing. Following the collection of *.fastq files, adapter trimming was done by flexbar60 (link),61 . RA3 (5’-TGGAATTCTCGGGTGCCAAGG-3’) and RA5 (5’-GTTCAGAGTTCTACAGTCCGACGATCNNNNNAGC-3’) adapter of Illumina TruSeq were selected as input for the flexbar run. For further sequence processing and statistical analysis, pyCRAC packages were used (https://git.ecdf.ed.ac.uk/sgrannem/pycrac ). pyFastqDuplicateRemover.py, a program, which removes duplicate from the flexbar trimmed files were used for sequence processing (pyCRAC package). Next, Bowtie2 v2.4.2 was used to align trimmed sequence on R64-1-1 reference genome (https://uswest.ensembl.org/Saccharomyces_cerevisiae/Info/Annotation ) and *.sam output format was produced for the downstream analyses. pyReadCounters.py program (from the same pyCRAC package) was used to calculate the RPKM values of genes.
3
Illumina Library Preparation from Phage DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysed phage DNA from each sample was amplified and readied for Illumina deep sequencing by performing two rounds of PCR, as previously described (Garrett et al., 2020 (link)). Each PCR reaction was performed using Q5 High-Fidelity 2X Master Mix. For the first round of PCR, 10uL of lysed phage was used as the template in a 25 uL reaction. For the second round of PCR, 2 uL of the round 1 PCR product was then used as the template in a 50 uL reaction, with primers that add dual indexing sequences on either side of the insert. PCR products were then cleaned using AMPure XP beads and eluted in 50 uL water. DNA concentrations were quantified via Quant-iT PicoGreen dsDNA Assay Kit. Equimolar amounts of DNA from the samples, along with 10X the amount of the input library samples, was pooled, gel purified, and the final library was quantified using the KAPA Library Quantification Kit. Pools were sequenced on an Illumina MiSeq with 1x125 bp single end reads using a custom sequencing primer.
4
CRISPR Sequencing Analysis Pipeline
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis for this paper was performed on 81 M raw reads from Illumina deep sequencing collected following the addition at four time points of the small molecule pomalidomide to a CRISPR library consisting of 48 well-characterized guide sequences. Sequencing reads were filtered to remove low-quality (Illumina average quality < 28) or unmapped reads and were genotyped. For each sequencing read representing a CRISPR-Cas9 cutting event, the cutting genotype was identified and categorized as an insertion or deletion event, and the overall fractions of insertions and deletions of all lengths were computed for two replicates at each time point. The analysis protocol was described previously.37 (link)
5
CRISPR-Cas9 Cutting Efficiency Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Analysis was performed on raw reads from Illumina deep sequencing. Sequencing reads were filtered to remove low-quality (Illumina average quality <28) or unmapped reads. For each sequencing read representing a CRISPR-Cas9 cutting event, the cutting genotype was identified and categorized as an insertion or deletion event; overall fractions of insertions and deletions of all lengths were then computed from the two replicates. This was carried out with a published analysis pipeline described in Shen et al. 12 (link). For indel and 1-bp insertion detection at native human genomic loci, Crispresso2 was used to carry out the alignment of NGS reads to the wild-type amplicon sequence, using default parameters and a Needleman Wunsch gap extended score of 0 for optimal alignment with visual inspection39 (link). Reads were determined to be modified or unmodified if a 1-bp insertion was located within 2 bp from each sgRNA cleavage (default quantification window for Cas9 Crispresso2).
6
Illumina-based tRNA Sequencing Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The RNA samples were outsourced for library construction and sequenced on the Illumina NextSeq500 System (KangChen Bio-tech, Shanghai, China). Briefly, the RNA samples were pretreated to remove some RNA modifications and were sequentially ligated to 3′ and 5′ small RNA adapters. The RNA was then reversed and amplified. Consequently, ~134-160 bp PCR amplified fragments were purified and used for the preparation of sequencing libraries. And finally, the libraries were sequenced by Illumina deep sequencing. The tRNA sequences of cytoplasmic were downloaded from GtRNAdb, and tRNA sequences of mitochondrial were predicted with tRNA scan-SE software. To generate the mature tRNA libraries, the predicted intronic sequences were moved. Meanwhile, we added an additional 3′-terminal “CCA” to each tRNA. We also included 40 nucleotides of flanking genomic sequence on either side of the original tRNA sequence in order to generate the precursor tRNA libraries. The generated adjusted p values lower than 0.05 were considered significant.
7
Custom oligo array and sgRNA library
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The custom oligonucleotide array (Supplementary Table 1 ) was synthesised by Custom Array Inc. Overlapping PCR was performed to incorporate NdeI and XbaI sites to the custom array for subsequent Gibson Assembly (NEB, Ipswich, MA). The PCR products were then cloned into pLX-sgRNA linearised with NdeI and XbaI. pLX-sgRNA was a kind gift from Eric Lander & David Sabatini (Addgene plasmid #50662).25 (link) The Gibson library reaction was transformed into XL10-Ultra competent cells. To maintain the complexity of the library, at least 20-fold coverage in library representation was recovered in the transformation and cultured in NYZM + broth for 7 h or until OD600 reached 0.8. Subsequently, deep sequencing (Illumina) was performed to validate the library complexity of the input plasmid and lentivirus pool.
8
Measuring Base Editing Frequencies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Base editing frequencies were measured either from liquid cultures 5 days after electroporation or in individual hematopoietic colonies grown in methylcellulose. The AAVS1 or FANCA exon 4 regions were amplified with AmpliTaq Gold 360 DNA Polymerase (Thermo Fisher Scientific) and corresponding primers using the following cycling conditions: 95 °C for 10 min; 40 cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 1 min; and 72 °C for 7 min. Primers used in these PCRs are listed in Supplemental Table 3 . Resulting PCR products were subjected to Sanger sequencing or illumina deep sequencing. For Sanger sequencing, PCR products were sequenced using Fw primers described in Supplemental Table 3 . For deep sequencing, PCR products were purified using the Zymo Research DNA Clean and Concentrator kit (#D4004), quantified using Qubit fluorometer (Thermo Fisher Scientific), and used for library construction for illumina platforms. The generated DNA fragments were sequenced by Genewiz with Illumina MiSeq Platform, using 250-bp paired-end sequencing reads. Frequencies of editing outcomes were quantified using CRISPResso2 software (quantification window center (-3) and size (-10); plot window size (20); base edit target A to G; batch mode).
9
Investigation of CRISPR-induced Indels in DM1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Investigation of indel types and distributions was conducted by Illumina deep sequencing of PCR amplicons generated from DM1 cells treated with an MOI 100 of lentiviral vectors expressing SaCas9 and sgRNA4-23 and from DMSXL TA muscles intramuscularly injected at 5–6 weeks of age with rAAV9 vectors expressing SaCas9 and sgRNA4-23. Untreated DM1 cells and TA muscle injected with PBS were used as controls. PCR amplicons of around 300 bp were generated by nested PCR. The first PCR was performed with a set of primers specific for the targets (F1-DMPK-3UTR and R2-DMPK-3UTR for the region containing the CTG repeat deletion; F1-DMPK-3UTR and R-DMPK bef CTG for the region surrounding the target of sgRNA 4; F-DMPK-149up-sgRNA23 and R2-DMPK-3UTR for the region surrounding the target of sgRNA 23). Amplicons generated from the first PCR served as the template for a second reaction, which was performed with a second set of primers annealing downstream of the first set and containing Illumina adaptors at the 5′ end of the sequence (see Table S2 ). Row deep sequencing reads from Illumina PE sequencing (IGATech) were merged using PEAR (version [v.]0.9.6, using: −n 50 –v 20 –q 20 –t 20)66 (link) and NEXTERA adaptor trimmed using cutadapt (v.1.18, using –e 0.2 −o 10 –q 20 –m 50).67 Merged sequences were then submitted to CRISPRESSO2 for INDELS discovery (using –ignore-substitutions–q 20).68 (link)
10
CRISPR Library Sequencing Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The analysis for this paper was performed on 81 M raw reads from Illumina deep sequencing collected following the addition of the small molecule dTAG-47 at four time points to a CRISPR library consisting of 48 well-characterized guide sequences. Sequencing reads were filtered to remove low-quality (Illumina average quality <28) or unmapped reads and were genotyped. For each sequencing read representing a CRISPR-Cas9 cutting event, the cutting genotype was identified and categorized as an insertion or deletion event, and overall fractions of insertions and deletions of all lengths were computed for two replicates at each different time point. The analysis protocol was described previously.38 (link)
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!