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| Status |
Public on Apr 03, 2024 |
| Title |
S_AJ_ALT_pos18 |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Blood
cell line: GM24385
cell type: B-Lymphocyte
genotype: WT
treatment: digested with sgRNA targeting ALT alelle
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
gDNA was obtained from Coriell
DNA DSB ends of nuclease-digested gDNA were repaired and 3’ adenylated using the NEBNext® Ultra™ II End Repair/dA-Tailing Module (NEB E7546) as described by the manufacturer with the following modification: Reaction volume was halved by adding half volume of the reagents. Labeling of DSB ends with BreakTag linker ligation was performed using the NEBNext® Ultra™ II Ligation Module (NEB E7595) according to manufacturer’s recommendation with the following modifications: Reaction volume was halved by adding half volume of reagents, and USER enzyme digestion step was omitted. BreakTag linker was used at a final concentration of 50 nM per sample. Labeled DNA was size-selected using DNA AMpure XP beads (0.7x volumes) in order to remove unligated linkers and eluted in nuclease-free H2O. Tagmentation with in-house Tn5 was performed in 10 mM Tris-HCl pH 7.5, 10 mM MgCl2, and 25% dimethylformamide (DMF, Sigma Aldrich 227056). Tagmentation reactions were assembled using 100-200 ng of DNA as input. Hyperactive Tn5 was used at a final concentration of 1.25 ng/µL per reaction. The tagmentation mix was then incubated at 55ᵒC for 5 minutes in a preheated thermocycler followed by termination with 0.2% SDS at room temperature for 5 minutes. Libraries were amplified with the NEBNext® Ultra™ II Q5® Master Mix (M0544). Amplified and barcoded samples were size-selected in order to remove PCR byproducts by performing two consecutive 0.5x volume right-tail + 0.35x volume left-tail size selection using DNA AMpure XP beads. Final libraries were quantified using Qubit dsDNA High Sensitivity Assay kit and fragment size distribution was assessed in a BioAnalyzer High Sensitivity DNA chip
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
CRISPResso version 2.2.12 called with following parameters: --amplicon_min_alignment_score 50 --quantification_window_size 10 --quantification_window_center -3 --exclude_bp_from_left 0 --exclude_bp_from_right 0 --ignore_substitutions --plot_window_size 20 --min_frequency_alleles_around_cut_to_plot 0
Supplementary files format and content: tab-separated file showing the number of modifications for positions in the quantification window of the amplicon. The first row shows the amplicon sequence in the quantification window, and successive rows show the number of reads with insertions (row 2), insertions_left (row 3), deletions (row 4), substitutions (row 5) and the sum of all modifications (row 6). Additionally, the last row shows the number of reads aligned.
Library strategy: BreakTag
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| Submission date |
Mar 17, 2023 |
| Last update date |
Apr 03, 2024 |
| Contact name |
Sergi Sayols |
| Organization name |
Institute of Molecular Biology, Mainz
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| Street address |
Ackermannweg 4
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| City |
Mainz |
| ZIP/Postal code |
55128 |
| Country |
Germany |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE223772 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag |
| GSE227604 |
Linking CRISPR/Cas9 double-strand break profiles to gene editing precision with BreakTag [indels] |
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| Relations |
| BioSample |
SAMN33798213 |
| SRA |
SRX19702067 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7103838_S_AJ_ALT_pos18.Quantification_window_modification_count_vectors.txt.gz |
345 b |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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