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Sample GSM7103838 Query DataSets for GSM7103838
Status Public on Apr 03, 2024
Title S_AJ_ALT_pos18
Sample type SRA
Source name Blood
Organism Homo sapiens
Characteristics tissue: Blood
cell line: GM24385
cell type: B-Lymphocyte
genotype: WT
treatment: digested with sgRNA targeting ALT alelle
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
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
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
Submission date Mar 17, 2023
Last update date Apr 03, 2024
Contact name Sergi Sayols
Organization name Institute of Molecular Biology, Mainz
Street address Ackermannweg 4
City Mainz
ZIP/Postal code 55128
Country Germany
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]
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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